Note: Descriptions are shown in the official language in which they were submitted.
WO 95/28421 ~ ~ PCT/US94/11690
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METHOD AND PRODUCT FOR REGULATING CELL
RESPONSIVENESS TO EXTERNAL SIGNALS
FIELD OF THE INVENTION
This invention relates to isolated nucleic acid
molecules encoding MEKK proteins, substantially pure MEKK
proteins, and products and methods for regulating signal
transduction in a cell.
SUMMARY OF THE INVENTION
The present invention relates to a substantially pure
MEKK protein capable of phosphorylating mammalian MEK
protein, in which the MEKK protein comprises a catalytic
domain. The present invention includes a substantially
pure MEKK protein capable of regulating signals initiated
from a growth factor receptor on the surface of a cell by
regulating the activity of MAPK protein, the ability to
regulate being divergent from Raf protein signal
regulation. In particular, the substantially pure MEKK
protein comprises at least a portion of an amino acid
sequence encoded by a nucleic acid sequence that is capable
of hybridizing under stringent conditions with a nucleic
acid molecule encoding an amino acid sequence including SEQ
ID N0:4, SEQ ID N0:6, SEQ ID N0:8 and SEQ ID NO:10. The
substantially pure MEKK protein capable of regulating the
activity of MAPK protein, said protein having an amino acid
sequence distinct from Raf protein.
- The present invention also includes a formulation
comprising at least one isolated protein having at least a
portion of an amino acid sequence encoded by a nucleic acid
sequence that is capable of hybridizing under stringent
conditions with a nucleic acid molecule encoding an amino
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acid sequence including SEQ ID NO: 2 , SEQ ID NO: 4 , SEQ ID
N0:6, SEQ ID N0:8 and SEQ ID NO:10.
One aspect of the present invention includes an
isolated nucleic acid molecule having a sequence encoding
a protein capable of phosphorylating mammalian MEK
independent of Raf protein and capable of regulating the
activity of MAPK protein. In particular, the present
invention includes an isolated nucleic acid molecule
capable of hybridizing under stringent conditions with a
nucleic acid sequence selected from the group consisting of
SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5, SEQ ID N0:7 and SEQ
ID N0:9.
Another aspect of the present invention includes a
recombinant molecule, comprising a nucleic acid molecule
capable of hybridizing under stringent conditions with a
nucleic acid sequence including SEQ ID NO:1, SEQ ID N0:3,
SEQ ID N0:5, SEQ ID N0:7 and SEQ ID N0:9, in which the
nucleic acid molecule is operatively linked to an
expression vector.
Yet another aspect of the present invention is a
recombinant cell transformed with a recombinant molecule,
comprising a nucleic acid molecule operatively linked to an
expression vector, the nucleic acid molecule comprising a
nucleic acid sequence capable of hybridizing under
stringent conditions with a nucleic acid sequence selected
from the group consisting of SEQ ID N0:1, SEQ ID N0:3, SEQ
ID N0:5, SEQ ID N0:7 and SEQ ID N0:9 (i.e., the nucleic
WO 95/28421 ? S ~ ~ PCT/US94/11690
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acid sequence shown in Table 1, Table 2, Table 3, Table 4
and Table 5).
The present invention also includes a method for
regulating the homeostasis of a cell comprising regulating
the activity of an MEKK-dependent pathway relative to the
activity of a Raf-dependent pathway in the cell. In
particular, the method comprises regulating the apoptosis
of the cell. Such a method is useful for the treatment of
a medical disorder. In particular, the method is useful
for inhibiting tumorigenesis and autoimmunity.
According to the present invention, the method for
treatment of a disease, comprises administering to a
patient an effective amount of a therapeutic compound
comprising at least one regulatory molecule including a
molecule capable of decreasing the activity of a Raf-
dependent pathway, a molecule capable of increasing the
activity of an MEKK-dependent pathway, and combinations
thereof, in which the effective amount comprises an amount
which results in the depletion of harmful cells involved in
the disease.
Also included in the present invention is a
therapeutic compound capable of regulating the activity of
an MEKK-dependent pathway in a cell identified by a
process, comprising: (a) contacting a cell with a putative
regulatory molecule; and (b) determining the ability of
the putative regulatory compound to regulate the activity
of an MEKK-dependent pathway in the cell by measuring the
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activation of at least one member of said MEKK-dependent
pathway.
One embodiment of the present invention includes a
substantially pure protein, in which the protein is
isolated using an antibody capable of selectively binding
to an MEKK protein capable of phosphorylating mammalian MEK
protein and capable of regulating the activity of MAPK
protein independent of Raf protein, the antibody capable of
being produced by a method comprising: (a) administering to
an animal an effective amount of a substantially pure MEKK
protein of the present invention; and (b) recovering an
antibody capable of selectively binding to the MEKK
protein.
Another embodiment of the present invention includes
an isolated antibody capable of selectively binding to an
MEKK protein, the antibody capable of being produced by a
method comprising administering to an animal an effective
amount of a substantially pure protein of the present
invention, and recovering an antibody capable of
selectively binding to the MEKK protein.
BACKGROUND OF THE INVENTION
Mitogen-activated protein kinase (MAPKs) (also called
extracellular signal-regulated kinases or ERKs) are rapidly
activated in response to ligand binding by both growth
factor receptors that are tyrosine kinases (such as the
epidermal growth factor (EGF) receptor) and receptors that
are coupled to heterotrimeric guanine nucleotide binding
proteins (G proteins) such as the thrombin receptor. The
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MAPKs appear to integrate multiple intracellular signals
transmitted by various second messengers. MAPKs
phosphorylate and regulate the activity of enzymes and
transcription factors including the EGF receptor, Rsk 90,
phospholipase A2, c-Myc, c-Jun and Elk-1/TCF. Although the
rapid activation of MAPKs by receptors that are tyrosine
kinases is dependent on Ras, G protein-mediated activation
of MAPK appears to occur through pathways dependent and
independent of Ras.
Complementation analysis of the pheromone-induced
signaling pathway in yeast has defined a protein kinase
system that controls the activity of Spkl and Fus3-Kssl,
the Schizosaccharomyces pombe and Saccharomyces cerevisiae
homologs of MAPK (see for example, B.R. Cairns et al.,
Genes and Dev. 6, 1305 (1992); B.J. Stevenson et al., Genes
and Dev. 6, 1293 (1992); S.A. Nadin-Davis et al., EMBO J.
7, 985 (1988); Y. Wang et al., Mol. Cell. Biol. 11, 3554
(1991). In S. cerevisiae, the protein kinase Ste7 is the
upstream regulator of Fus3-Kssl activity; the protein
kinase Stel1 regulates Ste7. The S. pombe gene products
Byri and Byr2 are homologous to Ste7 and Stell,
respectively. The MEK (MAPK Kinase or ERK Kinase) or MKK
(MAP Kinase kinase) enzymes are similar in sequence to Ste7
and Byri. The MEKs phosphorylate MAPKs on both tyrosine
and threonine residues which results in activation of MAPK.
The mammalian serine-threonine protein kinase Raf
phosphorylates and activates MEK, which leads to activation
of MAPK. Raf is activated in response to growth factor
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receptor tyrosine kinase activity and therefore Raf may
activate MAPK in response to stimulation of membrane-
associated tyrosine kinases. Raf is unrelated in sequence
to Stel1 and Byr2. Thus, Raf may represent a divergence in
mammalian cells from the pheromone-responsive protein
kinase system defined in yeast . Cel l and receptor specif is
differences in the regulation of MAPKs suggest that other
Raf independent regulators of mammalian MEKs exist.
Certain biological functions, such as growth and
differentiation, are tightly regulated by signal
transduction pathways within cells. Signal transduction
pathways maintain the balanced steady state functioning of
a cell. Disease states can arise when signal transduction
in a cell breaks down, thereby removing the tight control
that typically exists over cellular functions. For
example, tumors develop when regulation of cell growth is
disrupted enabling a clone of cells to expand indefinitely.
Because signal transduction networks regulate a multitude
of cellular functions depending upon the cell type, a wide
variety of diseases can result from abnormalities in such
networks. Devastating diseases such as cancer, autoimmune
diseases, allergic reactions, inflammation, neurological
disorders and hormone-related diseases can result from
abnormal signal transduction.
Despite a long-felt need to understand and discover
methods for regulating cells involved in various disease
states, the complexity of signal transduction pathways has
precluded the development of products and processes for
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regulating cellular function by manipulating signal
transduction pathways in a cell. As such, there remains a
need for products and processes that permit the
_ implementation of predictable controls of signal
transduction in cells, thus enabling the treatment of
various diseases that are caused by abnormal cellular
function.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 is a schematic representation of the signal
pathways of vertebrates and yeast.
Fig. 2 is a schematic representation of the dual MEKK
and Raf pathways divergent from Ras protein pathway.
Fig. 3A shows a Northern (RNA) blot of a single 7.8 kb
MEKK mRNA in several cell lines and mouse tissues.
Fig. 3B shows a Southern (DNA) blot of the MEKK gene.
Fig. 3C shows an immunoblot showing expression of the
78 kD and 50 kD forms of MEKK in rodent cell lines.
Fig. 4 shows immunprecipitates of MEKK protein using
MEKK antiserum.
Fig. 5 shows immunoblotting of MERK protein in
immunoprecipitates and cell lysates.
Fig. 6A shows the activation of MAPK in COS cells
- transfected with MEKK.
Fig. 6B is an immunoblot showing expression of MEKK in
cells either treated or not treated with EGF.
Fig. 7 shows the activation and phosphorylation of MEK
in COS cells transfected with MEKK.
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Fig. 8A shows the phosphorylation of MEK-1 by MEKK.
Fig. 8B shows the time course of phosphorylation of
MEK-1 by MEKK expressed in COS cells.
Fig. 8C is an immunoblot of MEKK overexpressed in COS
cells.
Fig. 9A shows the phosphorylation of MAPK by activated
MEK-1.
Fig. 9B shows phosphorylation of MEK-1 by
immunoprecipitated MEKK.
Fig. l0A shows the phosphorylation of MEK-1 by
activated Raf.
Fig. lOB shows the phosphorylation state of Raf
isolated from COS cells which are overexpressing MEKK and
have been treated with EGF.
Fig. 11 shows the relative ability of
immunoprecipitated MEKK and Raf-B to phosphorylate kinase
inactive MEK-1.
Fig. 12 shows a time course of EGF-stimulated MEKK and
Raf-B activation.
Fig. 13 shows that the immunodepletion of Raf-B from
MEKK immunoprecipitates has no effect on MEKK activity.
Fig. 14 shows that the immunodepletion of Raf-B from
MEKK immunoprecipitates decreases Raf-B activity.
Fig. 15 shows MEKK activity in FPLC Mono Q ion-
exchange column fractions of PC12 cell lysates.
Fig. 16 shows inhibition of MEKK and Raf-B activation
by dominant negative N~~RAS expression.
Fig. 17 shows activation of MEK protein by 98 kD MEKK.
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Fig. 18 shows inhibition of EGF activation of MEKK by
forskolin.
Fig. 19 shows improved MEKK activity by truncated MEKK
molecules.
Fig. 20 shows JNK activation by MEKK protein.
Fig. 21 shows regulation of c-Myc controlled
transcription and not CREB controlled transcription by MEKK
protein.
Fig. 22 is a schematic representation of MEKK
regulation of c-Myc controlled transcription.
Fig. 23 shows induction of p38 MAPK phosphorylation by
MEKK 3.
Fig. 24 shows induction of cellular apoptosis in Swiss
3T3 and REF52 cells by beauvericin.
Fig. 25 shows induction of cellular apoptosis in REF52
cells by MEKK.
Fig. 26 shows induction of cellular apoptosis in Swiss
3T3 and REF52 cells by MEKK.
Fig. 27 shows 3 representative microscopic views of
apoptotic REF52 cells expressing MEKK protein.
Fig. 28 shows 3 representative microscopic views of
apoptotic Swiss 3T3 cells expressing MEKK protein.
Fig. 29 shows similar stimulation of MAPK activity by
MEKK protein and Raf protein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel mitogen ERK
kinase kinase protein (MEKK) capable of regulating signal
WO 95/28421 ~ PCT/US94/11690
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transduction in cells. The present invention includes a
novel method for treating disease by regulating the
activity of cells involved in such disease. The present
invention is particularly advantageous in that the novel
product and method of the present invention is capable of
regulating a signal transduction pathway that can lead to
cellular apoptosis.
One embodiment of the present invention is an isolated
MEKK protein. According to the present invention, an
isolated protein is a protein that has been removed from
its natural milieu. An isolated MEKK protein can, for
example, be obtained from its natural source, be produced
using recombinant DNA technology, or be synthesized
chemically. As used herein, an isolated MEKK protein can
be a full-length MEKK protein or any homologue of such a
protein, such as an MEKK protein in which amino acids have
been deleted (e. g., a truncated version of the protein,
such as a peptide), inserted, inverted, substituted and/or
derivatized (e. g., by glycosylation, phosphorylation,
acetylation, myristoylation, prenylation, palmitoylation,
amidation and/or addition of glycosylphosphatidyl
inositol), wherein the modified protein is capable of
phosphorylating mitogen ERK kinase (MEK) and/or Jun ERK
kinase (JEK). A homologue of an MEKK protein is a protein
having an amino acid sequence that is sufficiently similar
to a natural MEKK protein amino acid sequence that a
nucleic acid sequence encoding the homologue is capable of
hybridizing under stringent conditions to (i.e., with) a
WO 95/28421 U ~ ~ PCT/US94/11690
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nucleic acid sequence encoding the natural MEKK protein
amino acid sequence. As used herein, stringent
hybridization conditions refer to standard hybridization
conditions under which nucleic acid molecules, including
oligonucleotides, are used to identify similar nucleic acid
molecules. Such standard conditions are disclosed, for
example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Labs Press, 1989. A
homologue of an MEKK protein also includes a protein having
an amino acid sequence that is sufficiently cross-reactive
such that the homologue has the ability to elicit an immune
response against at least one epitope of a naturally-
occurring MEKK protein.
The minimal size of a protein homologue of the present
invention is a size sufficient to be encoded by a nucleic
acid molecule capable of forming a stable hybrid with the
complementary sequence of a nucleic acid molecule encoding
the corresponding natural protein. As such, the size of
the nucleic acid molecule encoding such a protein homologue
is dependent on nucleic acid composition, percent homology
between the nucleic acid molecule and complementary
sequence, as well as upon hybridization conditions per se
(e. g., temperature, salt concentration, and formamide
concentration). The minimal size of such nucleic acid
molecules is typically at least about 12 to about 15
nucleotides in length if the nucleic acid molecules are GC-
rich and at least about 15 to about 17 bases in length if
they are AT-rich. As such, the minimal size of a nucleic
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acid molecule used to encode an MEKK protein homologue of
the present invention is from about 12 to about 18
nucleotides in length. There is no limit, other than a
practical limit, on the maximal size of such a nucleic acid
molecule in that the nucleic acid molecule can include a
portion of a gene, an entire gene, or multiple genes, or
portions thereof. Similarly, the minimal size of an MEKK
protein homologue of the present invention is from about 4
to about 6 amino acids in length, with preferred sizes
depending on whether a full-length, multivalent protein
(i.e., fusion protein having more than one domain each of
which has a function) , or a functional portion of such a
protein is desired.
MEKK protein homologues can be the result of allelic
variation of a natural gene encoding an MEKK protein. A
natural gene refers to the form of the gene found most
often in nature. MEKK protein homologues can be produced
using techniques known in the art including, but not
limited to, direct modifications to a gene encoding a
protein using, for example, classic or recombinant DNA
techniques to effect random or targeted mutagenesis. The
ability of an MEKK protein homologue to phosphorylate MEK
and/or JEK protein can be tested using techniques known to
those skilled in the art. Such techniques include
phosphorylation assays described in detail in the Examples
section.
In one embodiment, an MEKK protein of the present
invention is capable of regulating an MEKK-dependent
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pathway. According to the present invention, an MEKK-
dependent pathway refers generally to a pathway in which
MEKK protein regulates a pathway substantially independent
a of Raf, and a pathway in which MEKK protein regulation
converges with common members of a pathway involving Raf
protein, in particular, MEK protein (see Fig. 2). A
suitable MEKK-dependent pathway includes a pathway
involving MEKK protein and JEK protein, but not Raf
protein. One of skill in the art can determine that
regulation of a pathway by an MEKK protein is substantially
independent of Raf protein by comparing the ability of an
MEKK protein and a Raf protein to regulate the
phosphorylation of a downstream member of such pathway
using, for example, the general method described in Example
16. An MEKK protein regulates a pathway substantially
independently of Raf protein if the MEKK protein induces
phosphorylation of a member of the pathway downstream of
MEKK (e.g., proteins including JEK, JNK, Jun and/or ATF-2)
by an amount significantly greater than that seen when Raf
protein is utilized. For example, MEKK induction of
phosphorylation of JNK is preferably at least about 10-
fold, more preferably at least about 20-fold and even more
preferably at least about 30-fold, greater phosphorylation
of JNK protein than the phosphorylation induced when using
Raf protein. If MEKK induction of phosphorylation is
similar to Raf protein induction of phosphorylation, then
one of skill in the art can conclude that regulation of a
pathway by an MEKK protein includes members of a signal
WO 95/28421 O ~ ~ PCT/LJS94/1169(1
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transduction pathway that could also include Raf protein.
For example, MEKK induction of phosphorylation of MAPK is
of a similar magnitude as induction of phosphorylation with
Raf protein.
A "Raf-dependent pathway" can refer to a signal
transduction pathway in which Raf protein regulates a
signal transduction pathway substantially independently of
MEKK protein, and a pathway in which Raf protein regulation
converges with common members of a pathway involving MEKK
protein. The independence of regulation of a pathway by a
Raf protein from regulation of a pathway by an MEKK protein
can be determined using methods similar to those used to
determine MEKK independence.
In another embodiment, an MEKK protein is capable of
regulating the activity of signal transduction proteins
including, but not limited to, mitogen ERK kinase (MEK),
mitogen activated protein kinase (MAPK), transcription
control factor (TCF), Ets-like-1 transcription factor (Elk
1), Jun ERK kinase (JEK), Jun kinase (JNK), stress
activated MAPK proteins, Jun, activating transcription
factor-2 (ATF-2) and/or Myc protein. As used herein, the
"activity" of a protein can be directly correlated with the
phosphorylation state of the protein and/or the ability of
the protein to perform a particular function (e. g.,
phosphorylate another protein or regulate transcription).
Preferred MEK proteins regulated by an MEKK protein of the
present invention include MEK-1 and/or MEK-2. Preferred
MAPK proteins regulated by an MEKK protein of the present
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invention include p38 MAPK, p42 MAPK and/or p44 MAPK. A
preferred MEKK protein that is capable of phosphorylating
p38 MAPK protein includes a protein encoded by the nucleic
acid sequence represented by SEQ ID N0:5 with a protein
having the amino acid sequence represented by SEQ ID N0:7
being more preferred. Preferred stress activated MAPK
proteins regulated by an MEKK protein of the present
invention include Jun kinase (JNK), stress activated MAPK-a
and/or stress activated MAPK-B. An MEKK protein of the
l0 present invention is capable of increasing the activity of
an MEK protein over basal levels of MEK (i.e., levels found
in nature when not stimulated). For example, an MEKK
protein is preferably capable of increasing the
phosphorylation of an MEK protein by at least about 2-fold,
more preferably at least about 3-fold, and even more
preferably at least about 4-fold over basal levels when
measured under conditions described in Example 9.
A preferred MEKK protein of the present invention is
also capable of increasing the activity of an MAPK protein
2o over basal levels of MAPK (i.e., levels found in nature
when not stimulated). For example, an MEKK protein of the
present invention is preferably capable of increasing MAPK
activity at least about 2-fold, more preferably at least
about 3-fold, and even more preferably at least about 4-
fold over basal activity when measured under the conditions
described in Example 3.
Moreover, an MEKK protein of the present invention is
capable of increasing the activity of a JNK protein. JNK
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regulates the activity of the transcription factor JUN
which is involved in controlling the Qrowth and
differentiation of different cell types, such as T cells,
neural cells or fibroblasts. JNK shows structural and
regulatory homologies with MAPK. For example, an MEKK
protein of the present invention is preferably capable of
inducing the phosphorylation of JNK protein at least about
30 times more than Raf, more preferably at least about 40
times more than Raf, and even more preferably at least
l0 about 50 times more than Raf, when measured under
conditions described in Example 16.
A preferred MEKK protein of the present invention is
additionally capable of inducing the phosphorylation of a
c-Myc transcriptional transactivation domain protein in
such a manner that the phosphorylated transcriptional
transactivation domain of c-Myc is capable of regulating
gene transcription. The ability of an MEKK protein to
regulate phosphorylation of a c-Myc transcriptional
transactivation domain protein exceeds the ability of Raf
protein or cyclic AMP-dependent protein kinase to regulate
a c-Myc protein. For example, an MEKK protein of the
present invention is preferably capable of inducing
luciferase gene transcription by phosphorylated c-Myc
transcriptional transctivation domain protein at least
about 25-fold, more preferably at least about 35-fold, and
even more preferably at least about 45-fold, over Raf
induction when measured under the conditions described in
Example 17.
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Another aspect of the present invention relates to the
ability of MEKK activity to be stimulated by growth factors
including, but not limited to, epidermal growth factor
(EGF), neuronal growth factor (NGF), tumor necrosis factor
(TNF), CSA, interleukin-8 (IL-8), monocyte chemotactic
protein 1 (MIPla), monocyte chemoattractant protein 1 (MCP-
1), platelet activating factor (PAF), N-Formyl-methionyl-
leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4R), gastrin
releasing peptide (GRP), IgE, major histocompatibility
l0 protein (MHC), peptide, superantigen, antigen, vasopressin,
thrombin, bradykinin and acetylcholine. In addition, the
activity of an MEKK protein of the present invention is
capable of being stimulated by compounds including phorbol
esters such as TPA. A preferred MEKK protein is also
capable of being stimulated by EGF, NGF and TNF (especially
TNFa).
Preferably, the activity of an MEKK protein of the
present invention is capable of being stimulated at least
2-fold over basal levels (i.e., levels found in nature when
not stimulated) , more preferably at least about 4-fold over
basal levels and even more preferably at least about 6-fold
over basal levels, when a cell producing the MEKK protein
is contacted with EGF under the conditions described in
Example 3.
Similarly, the activity of an MEKK protein of the
present invention is capable of being stimulated at least
1-fold over basal levels, more preferably at least about 2-
fold over basal levels and even more preferably at least
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about 3-fold over basal levels by NGF stimulation, when a
cell producing the MEKK protein is contacted with NGF under
the conditions described in Example 9.
Preferably, an MEKK protein of the present invention
is capable of being stimulated at least 0.5-fold over basal
levels, more preferably at least about 1-fold over basal
levels and even more preferably at least about 2-fold over
basal levels by TPA stimulation when a cell producing the
MEKK protein is contacted with TPA under the conditions
described in Example 9.
TNF is capable of regulating cell death and other
functions in different cell types. The present inventor
discovered that MEKK stimulation by TNF is independent of
Raf. Similarly, the present inventor is the first to
appreciate that an MEKK protein can be directly stimulated
by ultraviolet light (W) damage of cells while a Raf
dependent pathway cannot. Therefore, both TNF and UV
stimulate MEKK activity without substantially activating
Raf . In addition, both UV and TNF activation of MEKK is
Ras dependent.
Another aspect of the present invention is the
recognition that an MEKK protein of the present invention
is capable of regulating the apoptosis of a cell, an
ability not shared by Raf protein. As used herein,
apoptosis refers to the form of cell death that comprises:
progressive contraction of cell volume with the
preservation of the integrity of cytoplasmic organelles;
condensation of chromatin, as viewed by light or electron
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microscopy; and DNA cleavage, as determined by centrifuged
sedimentation assays. Cell death occurs when the membrane
integrity of the cell is lost and cell lysis occurs.
Apoptosis differs from necrosis in which cells swell and
eventually rupture.
A preferred MEKK protein of the present invention is
capable of inducing the apoptosis of cells, such that the
cells have characteristics substantially similar to
cytoplasmic shrinkage and/or nuclear condensation as shown
in Figs. 24, 25, 26, 27 and 28. The apoptotic cells in
Figs. 24 through 28 were obtained when cells were
microinjected with expression plasmids encoding MEKK
protein. Injected cells were identified using anti-8-Gal
antibody and the DNA of the cells were stained with
propidium iodide. Cytoplasmic organization was monitored
using an anti-tubulin antibody. The cells were then imaged
by differential fluorescent imaging microscopy using
techniques standard in the art. The cells demonstrated
apoptosis by displaying a morphology having cytoplasmic
shrinkage and nuclear condensation.
A schematic representation of the cell growth
regulatory signal transduction pathway that is MEKK
dependent is shown in Fig. 2. An MEKK protein of the
present invention is capable of regulating the activity of
JEK protein, JNK protein, Jun protein and/or ATF-2 protein,
and Myc protein, such regulation being substantially, if
not entirely, independent of Raf protein. Such Raf-
independent regulation can regulate the growth
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characteristics of a cell, including the apoptosis of a
cell. In addition, an MEKK protein of the present
invention is capable of regulating the activity of MEK
protein, which is also capable of being regulated by Raf
protein. As such, an MEKK protein of the present invention
is capable of regulating the activity of MAPK protein and
members of the Ets family of transcription factors, such as
TCF protein, also referred to as Elk-1 protein.
Referring to Fig. 2, an MEKK protein of the present
invention is capable of being activated by a variety of
growth factors capable of activating Ras protein. In
addition, an MEKK protein is capable of activating JNK
protein which is also activated by Ras protein, but is not
activated by Raf protein. As such, an MEKK protein of the
present invention comprises a protein kinase at a
divergence point in a signal transduction pathway initiated
by different cell surface receptors. An MEKK protein is
also capable of being regulated by TNF protein independent
of Raf, thereby indicating an association of MEKK protein
to a novel signal transduction pathway which is independent
of Ras protein and Raf protein. Thus, an MEKK protein is
capable of performing numerous unique functions independent
of or by-passing Raf protein in one or more signal
transduction pathways. An MEKK protein is capable of
regulating the activity of MEK and/or JEK activity. As
such, an MEKK protein is capable of regulating the activity
of members of a signal transduction pathway that does not
substantially include Raf activity. Such members include,
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but are not limited to, JNK, Jun, ATF and Myc protein. In
addition, an MEKK protein is capable of regulating the
members of a signal transduction pathway that does involve
Raf, such members including, but are not limited to, MEK,
MAPK and TCF. An MEKK protein of the present invention is
thus capable of regulating the apoptosis of a cell
independent of significant involvement by Raf protein.
In addition to the numerous functional characteristics
of an MEKK protein, an MEKK protein of the present
l0 invention comprises numerous unique structural
characteristics. For example, in one embodiment, an MEKK
protein of the present invention includes at least one of
two different structural domains having particular
functional characteristics. Such structural domains
include an NHZ-terminal regulatory domain that serves to
regulate a second structural domain comprising a COOH-
terminal protein kinase catalytic domain that is capable of
phosphorylating an MEK protein and/or JEK protein.
According to the present invention, an MEKK protein of
2o the present invention includes a full-length MEKK protein,
as well as at least a portion of an MEKK protein capable of
performing at least one of the functions defined above.
The phrase "at least a portion of an MEKK protein" refers
to a portion of an MEKK protein encoded by a nucleic acid
molecule that is capable of hybridizing, under stringent
conditions, with a nucleic acid encoding a full-length MEKK
protein of the present invention. Preferred portions of
MEKK proteins are useful for regulating apoptosis in a
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cell. Additional preferred portions have activities useful
for regulating MEKK kinase activity. Suitable sizes for
portions of an MEKK protein of the present invention are as
disclosed for MEKK protein homologues of the present
invention.
In another embodiment, an MEKK protein of the present
invention includes at least a portion of an MEKK protein
having molecular weights ranging from about 70 kD to about
250 kD as determined by Tris-glycine SDS-PAGE, preferably
using an 8% polyacrylamide SDS gel (SDS-PAGE) and resolved
using methods standard in the art. A preferred MEKK
protein has a molecular weight ranging from about 75 kD to
about 225 kD and even more preferably from about 80 kD to
about 200 kD.
In yet another embodiment, an MEKK protein of the
present invention comprises at least a portion of an MEKK
protein encoded by an mRNA (messenger ribonucleic acid)
ranging from about 3.5 kb to about 12.0 kb, more preferably
ranging from about 4.0 kb to about 11.0 kb, and even more
preferably ranging from about 4.5 kb to about 10.0 kb.
Particularly preferred MEKK proteins comprise at least a
portion of an MEI~ protein encoded by an mRNA having a size
ranging from about 4.5 kb to about 5.0 kb, a size ranging
from about 6.0 kb to about 6.5 kb, a size of about 7.0 kb,
or a size ranging from about 8.0 kb to about 10.0 kb.
In another embodiment, an NHZ-terminal regulatory
domain of the present invention includes an NH2-terminal
comprising about 400 amino acids having at least about 10%
WO 95/28421 ~ ~ PCT/US94/11690
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serine and/or threonine residues, more preferably about 400
amino acids having at least about 15% serine and/or
threonine residues, and even more preferably about 400
amino acids having at least about 20% serine and/or
threonine residues.
A preferred an NHZ-terminal regulatory domain of the
present invention includes an NH2-terminal comprising about
360 amino acids having at least about 10% serine and/or
threonine residues, more preferably about 360 amino acids
having at least about 15% serine and/or threonine residues,
and even more preferably about 360 amino acids having at
least about 20% serine and/or threonine residues.
Another preferred an NHZ-terminal regulatory domain of
the present invention includes an NH2-terminal comprising
about 370 amino acids having at least about 10% serine
and/or threonine residues, more preferably about 370 amino
acids having at least about 15% serine and/or threonine
residues, and even more preferably about 370 amino acids
having at least about 20% serine and/or threonine residues.
In one embodiment, an MEKK protein of the present
invention is devoid of SH2 and SH3 domains.
In another embodiment, an MEKK protein of the present
invention includes at least a portion of an MEIZK protein
homologue preferably having at least about 50%, more
preferably at least about 75%, and even more preferably at
least about 85% amino acid homology (identity within
comparable regions) with the kinase catalytic domain of a
naturally occurring MEKK protein. Another MEKK protein of
21~6~26
WO 95/28421 PCT/US94/11690
-24-
the present invention also includes at least a portion of
an MEKI~ homologue of the present invention has at least
about 10%, more preferably at least about 20%, and even
more preferably at least about 30% amino acid homology with
the NH2-terminal regulatory domain of an MEKK protein of a
naturally occurring MEKK protein.
The sequences comprising the catalytic domain of an
MEKK protein are involved in phosphotransferase activity,
and therefore display a relatively conserved amino acid
sequence. The NHz-terminal regulatory domain of an MEKK
protein, however, can be substantially divergent. The lack
of significant homology between MEKK protein Nliz-terminal
regulatory domains is related to the regulation of each of
such domains by different upstream regulatory proteins.
For example, an MEKK protein can be regulated by the
protein Ras, while others can be regulated independent of
Ras. In addition, some MEKK proteins can be regulated by
the growth factor TNFa, while others cannot. As such, the
NHZ-terminal regulatory domain of an MEKK protein provides
selectivity for upstream signal transduction regulation,
while the catalytic domain provides for MEKK substrate
selectivity function.
A preferred MEKK homologue has at least about 50%,
more preferably at least about 75% and even more preferably
at least about 85% amino acid homology with the kinase
catalytic domain of an MEKK protein having an amino acid
sequence represented by SEQ ID NO: 2 , SEQ ID NO: 4 , SEQ ID
N0:6, SEQ ID N0:8 or SEQ ID NO:10. Another preferred MEKK
WO 95/28421 ~ i J PCT/US94111690
-25-
homologue has at least about 10%, more preferably at least
about 20% and even more preferably at least about 30% amino
acid homology with the NHZ-terminal regulatory domain of an
MEKK protein having an amino acid sequence represented by
SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8 or SEQ
ID NO:10.
In a preferred embodiment, an MEKK protein of the
present invention includes at least a portion of an MEKK
protein homologue of the present invention that is encoded
by a nucleic acid molecule having at least about 50%, more
preferably at least about 75%, and even more preferably at
least about 85% homology with a nucleic acid molecule
encoding the kinase catalytic domain of an MEKK protein.
Another preferred MEKK protein homologue is encoded by a
nucleic acid molecule having at least about 10%, more
preferably at least about 20%, and even more preferably at
least about 30% homology with a nucleic acid molecule
encoding the NHZ-terminal regulatory domain of an MEKK
protein.
Still another preferred MEKK homologue is encoded by
a nucleic acid molecule having at least about 50%, more
preferably at least about 75% and even more preferably at
least about 85% amino acid homology With the kinase
catalytic domain of an MEKK protein encoded by a nucleic
acid sequence represented by SEQ ID NO:l, SEQ ID N0:3, SEQ
ID N0:5, SEQ ID N0:7 or SEQ ID N0:9. An MEKK homologue
also includes those encoded by a nucleic acid molecule
having at least about 10%, more preferably at least about
21 ~36~~6
WO 95/28421 . PCT/L1S94/11690
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20% and even more preferably at least about 30% amino acid
homology with the NH2-terminal regulatory domain of an,MEKK
protein encoded by a nucleic acid sequence represented by
SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5, SEQ ID N0:7 or SEQ
ID N0:9.
An MEKK protein of the present invention, referred to
here as MEKK 1, includes an MEKK protein having (i.e.,
including) at least a portion of the nucleic acid and/or an
amino acid sequence shown in Table 1 and represented by SEQ
ID NO:1 and SEQ ID N0:2, respectively.
Table 1.
TACACTCCTT GCCACAGTCT GGCAGAAAGAAGAGACTCCT CCGGCCAGTT60
ATCAAACTTC
GTAGACACTA TCCTTGTCAA GTGTGCAGATCACGAGTCAG CTGTCCATAT120
CCAACAGCCG
CTACAGTGCT GGAACTCTGC AAGGGCCAAGGGCGGTTGGG AGAGAMTAC180
CAGGAGAGCT
1 TTAMGCTGG GTCCATCGGG GTTGGTGGTGCTTMGTTGT ATCCTTGGAA240
5 TCGATTACGT
ACCAAGCTGA ATCAAACAAC TGGCMGAACCCTCTGTCTT ATAGACAGGT300
TGCTGGGTCG
TGCTGTTGGA ATTTCCTGCT GAATTCTATCCAGTACTGAT GTCTCACMG360
CTCATATTGT
CTGAGCCTGT TGAAATCAGG TACMGAAGCCTTAACCTTT GCCTTGCMT420
TGCTCTCCCT
CCATTGACAA TTCCCACTCG ATGGTTGGCAG11GGATATAT 480
AGCTCTCTCG CTGAGCTCTG
2 CCAGG ATG GTG ACC GCA GTG 527
O CCC GCT GTG TTT TCC MG
CTG GTA ACC
llet Val Thr Ala Yal Pro
Ala Val Phe Ser Lys Leu
Val Thr
1 5 10
ATG CTT AAT GCT TCT GGC AGG ATG CGC 575
TCC ACC CAC TTC ACC CGG CGT
llet Leu Asn Ale Ser Gly Arg llet Arg
Ser Thr His Phe Thr Arg Arg
2 15 20 25 30
5
CTG ATG GCT ATC GCG GAT GAG GTC ATC 623
GAG GTA GAA ATT GCC CAG CTG
Leu llet Ala Ile Ala Asp Glu Val Ile
Glu Val Glu Ile Ala Gln Leu
35 40 45
GGT GTG GAG GAC ACT GTG AGC TTA CAG 671
GAT GGG CAT CAG GAC GCC GTG
3 Gly Val Glu Asp Thr Val Ser Leu Gln
0 Asp Gly His Gln Asp Ala Val
50 55 60
GCC CCC ACC AGC TGT CTA GAG CAC ACA 719
GAA AAC AGC TCC CTT GTC CAT
Ala Pro Thr Ser Cys Leu Glu His Thr
Glu Asn Ser Ser Leu Val His
65 70 75
3 AGA GAG AAA ACT GGA AM GGA AGA CTG AGT 767
5 CTA AGT GCT ACG GCC AGC
Arg Gtu Lys Thr Gly Lys Arg Leu Ser
Gly Leu Ser Ala Thr Ala Ser
80 85 90
TCG GAG GAC ATT TCT GAC TCT GTA GGA 815
AGA CTG GCC GGC GTC CTT CCC
Ser Glu Asp Ile Ser Asp Ser Yal Gly
Arg Leu Ala Gly Val Leu Pro
4 95 100 105 110
0
AGC TCA ACA ACA ACA GM CAA GTT CM ACA AM 863
CCA AAG CCA GCG GGC
Ser Ser Thr Thr Thr Glu Yal Gln Thr
Gln Pro Lys Pro Ala Lys Gly
115 120 125
AGA CCC CAC AGT CAG TGT TTG TCT CAT 911
TTG AAC TCC TCC CCT GCT CAA
4 Arg Pro His Ser Gln Cys Leu Ser His
5 Leu Asn Ser Ser Pro Ala Gln
130 135 140
21~~5~6
WO 95/28421 PCT/US94/11690
-27-
TTA ATG TTC CCA GCA CCA TCA GCC CCT TGT 959
TCC TCT GCC CCG TCT GTC
Leu Met Phe Pro Ala Pro Ser Ala Pro Cys
Ser Ser Ala Pro Ser Val
145 150 155
CCA GAT ATT TCT MG CAC AGA CCC CAG GCA 1007
TTT GTT CCC TGC AM ATA
Pro Asp Ile Ser Lys His Arg Pro Gln Ala
Phe Val Pro Cys Lys Ile
160 165 170
CCT TCC GCA TCT CCT CAG ACA CAG CGC MG 1055
TTC TCT CTA CM TTC CAG
Pro Ser Ala Ser Pro Gln Thr Gln Arg Lys
Phe Ser Leu Gln Phe Gln
175 180 185 190
Z AGG MC TGC TCT GM CAC CGA GAC TCA GAC 1103
O CAG CTC TCC CCA GTC TTC
Arg Asn Cys Ser Glu His Arg Asp Ser Asp
Gln leu Ser Pro Val Phe
195 200 205
ACT CAG TCA AGA CCC CCA CCC TCC AGT MC 1151
ATA CAC AGG CCA MG CCA
Thr Gln Ser Arg Pro Pro Pro Ser Ser Asn
Ile His Arg Pro Lys Pro
210 215 220
TCC CGA CCC GTT CCG GGC AGT ACA AGC AM 1199
CTA GGG GAC GCC ACA AM
Ser Arg Pro Yal Pro Gly Ser Thr Ser Lys
Leu Gly Asp Ala Thr Lys
225 230 235
AGT AGC ATG ACA CTT GAT CTG GGC AGT GCT 1247
TCC AGG TGT GAC GAC AGC
2 Ser Ser llet Thr Leu Asp Leu Gly Ser Ala
0 Ser Arg Cys Asp Asp Ser
240 245 250
TTT GGC GGC GGC GGC MC AGT GGC MC GCC 1295
GTC ATA CCC AGC GAC G11G
Phe Gly Gly Gly Gly Asn Ser Gly Asn Ala
Yal Ile Pro Ser Asp Glu
255 260 265 270
2 ACA GTG TTC ACG CCG GTG GAG GAC MG TGC 1343
5 AGG TTA GAT GTG MC ACC
Thr Val Phe Thr Pro Yal Glu Asp Lys Cys
Arg leu Asp Val Asn Thr
275 280 285
GAG CTC MC TCC AGC ATC GAG GAC CTT CTT 1391
GM GCA TCC ATG CCT TCA
Glu Leu Asn Ser Ser Ile Glu Asp Leu Leu
Glu Ala Ser Met Pro Ser
3 290 295 300
0
AGT GAC ACG ACA GTC ACT TTC MG TCC GAA 1439
GTC GCC GTC CTC TCT CCG
Ser Asp Thr Thr Yal Thr Phe Lys Ser Glu
Val Ala Val Leu Ser Pro
305 310 315
GM MG GCC GM MT GAC GAC ACC TAC AM GAC 1487
GAC GTC MT CAT MT
3 Glu Lys Ala Glu Asn Asp Asp Thr Tyr Lys
5 Asp Asp Val Asn His Asn
320 325 330
CM MG TGC AM GM MG ATG GM GCT GM GAG GAG 1535
GAG GCT TTA GCG
Gln Lys Cys Lys Glu Lys llet Glu Ala Glu
Glu Glu Glu Ala Leu Ala
335 340 345 350
4 ATC GCC ATG GCG ATG TCA GCG TCT CAG GAT 1583
O GCC CTC CCC ATC GTC CCT
Ile Ala Met Ala Net Ser Ala Ser Gln Asp
Ala Leu Pro Ile Val Pro
355 360 365
CAG CTG CAG GTG GM MT GGA GM GAT ATT ATC 1631
ATC ATT CAG CAG GAC
Gln Leu Gln Val Glu Asn Gly Glu Asp Ile
Ile Ile Ile Gln Gln Asp
4 370 375 380
5
ACA CCA GM ACT CTT CCA GGA CAT ACC AM 1679
GCG AM CAG CCT TAC AGA
Thr Pro Glu Thr Leu Pro Gly His Thr Lys
Ala Lys Gln Pro Tyr Arg
385 390 395
GM GAC GCT GAG TGG CTG AM GGC CAG CAG 1727
ATA GGC CTC GGA GCA TTT
5 Glu Asp Ala Glu Trp Leu Lys Gly Gln Gln
0 Ile Gly Leu Gly Ala Phe
400 405 410
TCT TCC TGT TAC CM GCA CAG GAT GTG GGG 1775
ACT GGG ACT TTA ATG GCT
Ser Ser Cys Tyr Gln Ala Gln Asp Val Gly
Thr Gly Thr Leu llet Ala
415 420 425 430
5 GTG AM CAG GTG ACG TAC GTC AGA MC ACA 1823
5 TCC TCC GAG CAG GAG GAG
Val Lys Gln Vat Thr Tyr Val Arg Asn Thr
Ser Ser Glu Gln Glu Glu
218652.6
WO 95!28421 PCT/ITS94/11690
-28-
435 440 445
GTG GTG GAA GCG TTG AGG GAA GAG ATC CGG 1871
ATG ATG GGT CAC CTC MC
Val Val Glu Ala Leu Arg Glu Glu Ile.Arg
Met Met Gly His Leu Asn
450 455 460
CAT CCA MC ATC ATC CGG ATG CTG GGG GCC 1919
ACG TGC GAG MG AGC MC
His Pro Asn lle Ile Arg !let Leu Gly Ala
Thr Cys Glu Lys Ser Asn
465 470 475
TAC MC CTC TTC ATT GAG TGG ATG GCG GGA 1967
GGA TCT GTG GCT CAC CTC
Tyr Asn Leu Phe Ile Glu Trp Met Ala Gly
Gly Ser Yal Ala His Leu
480 485 490
TTG AGT MA TAC GGA GCT TTC MG GAG TCA GTC 2015
GTC ATT MC TAC ACT
Leu Ser Lys Tyr Gly Ala Phe Lys Glu Ser
Val Val Ile Asn Tyr Thr
495 500 505 510
GAG CAG TTA CTG CGT GGC CTT TCC TAT CTC 2063
CAC GAG MC CAG ATC ATT
1 Glu Gtn Leu Leu Arg Gly Leu Ser Tyr Leu
5 Nis Glu Asn Gln Ile 1le
515 520 525
CAC AGA GAC GTC MA GGT GCC MC CTG CTC ATT 2111
GAC AGC ACC GGT CAG
His Arg Asp Val Lys Gly Ala Asn Leu Leu
Ile Asp Ser Thr Gly Gln
530 535 540
2 AGG CTG AGA ATT GCA GAC TTT GGA GCT GCT 2159
O GCC AGG TTG GCA TCA MA
Arg Leu Arg Ile Ala Asp Phe Gly Ala Ala
Ala Arg Leu Ala Ser Lys
545 550 555
GGA ACC GGT GCA GGA GAG TTC CAG GGA CAG 2207
TTA CTG GGG ACA ATT GCA
Gly Thr Gly Ala Gly Glu Phe Gln Gly Gln
Leu Leu Gly Thr Ile Ala
2 560 565 570
5
TTC ATG GCG CCT GAG GTC CTA AGA GGT CAG 2255
CAG TAT GGT AGG AGC TGT
Phe Met Ala Pro Glu Val Leu Arg Gly Gln
Gln Tyr Gly Arg Ser Cys
575 580 585 590
GAT GTA TGG AGT GTT GGC TGC GCC ATT ATA 2303
GAA ATG GCT TGT GCA AAA
3 Asp Val Trp Ser Val Gly Cys Ala 1le Ile
0 Glu Met Ala Cys Ala Lys
595 600 605
CCA CCT TGG MT GCA GM MA CAC TCC MT CAT 2351
CTC GCC TTG ATA TTT
Pro Pro Trp Asn Ala Glu Lys Nis Ser Asn
His Leu Ala Leu Ile Phe
610 615 620
3 MG ATT GCT AGC GCA ACT ACT GCA CCG TCC 2399
5 ATC CCG TCA CAC CTG TCC
Lys Ile Ala Ser Ala Thr Thr Ala Pro Ser
Ile Pro Ser His Leu Ser
625 630 635
CCG GGT CTG CGC GAC GTG GCC GTG CGC TGC 2447
TTA GAA CTT CAG CCT CAG
Pro Gly Leu Arg Asp Val Ala Val Arg Cys
Leu Glu Leu Gln Pro Gln
4 640 645 650
0
GAC CGG CCT CCG TCC AGA GAG CTG CTG MA CAT CCG GTC TTC CGT ACC 2495
Asp Arg Pro Pro Ser Arg Glu Leu Leu Lys His Pro Val Phe Arg Thr
655 660 665 670
ACG TGG TAGTTMTTG TTCAGATCAG CTCTMTGGA GACAGGATAT CGMCCGGGA 2551
45 Thr Trp
GAGAGAAMG AGMCTTGTG GGCGACCATG CCGCTMCCG CAGCCCTCAC GCCACTGAAC 2611
AGCCAGAAAC GGGGCCAGCG GGGAACCGTA CCTMGCATG TGATTGACM ATCATGACCT 2671
GTACCTMGC TCGATATGCA GACATCTACA GCTCGTGCAG GMCTGCACA CCGTGCCTTT 2731
5 O CACAGGACTGGCTCTGGGGG CGATGGAGTT 2791
ACCAGGMGG TGCATGACTA
MGAACAGAA
GCATAMTTT ATTTTTGGAG GCTMTCAGT ACATCMCAT2851
CACTTTTTCA ATTACCATGT
GCCCGCCACA TTTCAMCTC AGACTGTCCCAGATGTCMG TTTGAGTTTG2911
ATCCACTGTG
TTTGCAGTTC CCTCAGCTTG TGGTGTTTTG MATGTG11TG2971
CTGGTMTTG TTTTCGATGC
TMTATTCTT ATTTTCTTTG GGACTGAAM TMTTATTTT3031
GATCAMGCT TTGTACTGTG
5 5 TGTGTTTTTAATGTTATTTG TGTAMTMC GTCTACTGCTGTTTATTCCA3091
GTACTCGMT
GTTTCTACTA CCTCAGGTGT TTTCTTCTAC TCTCAGAATG3151
CCTATAGATT CAMGTTCAC
AMTTCTACG TGCTGTGTGA TMGACTTCC GGCTMCTCC3211
CTATGACTCC AGGGCTTMG
TATTAGCACC TTACTATGTA ACAAAAAAAA 3260
AGCAMTGCT AAAAAAAAA
WO 95/28421 2_ 18 6 5 ? ~ pCT~S94/11690
-29-
An MEKK protein of the present invention, referred to
here as MEKK 2, includes an MEKK protein having at least a
portion of the nucleic acid and/or amino acid sequence
shown in Table 2 and represented by SEQ ID N0:3 and SEQ ID
N0:4, respectively.
Table 2.
GGTGGCGGCC GCTCTAGMC CCCGGGCTGC AGGMTTCGG 60
TAGTGGATCC CACGAGGGAC
GATCCAGCGG CAGAGTCGCC CGCTGCTTCT CCGGTCGGCG120
GCTTCCGCTT ACGCGGGCCC
GGGGCTTCCT TTTCATCGGC CCGCGGGCCC CGGGGCTGCA180
CCAGCTTATT GCTACCCAGA
Z AGCGGCGMG AGGCCCTGGG GCTGTCCCAT GTGMGCAGG 240
O CTGCGCGCCC TTGGGCCTGG
TCCCCGGCCC GTGCCCGGTT CTTCAGGCCT CAGGGACCCC300
GTCTGCGGCC CGCGAGGCGC
TGCTCCTGGG GGGCGCGGTG CGGGGGCGGA GGGGCCAGCT360
ACAGGCCGTG CGGTGGCCTC
CTCTCGGCCC TCGCGTCCGC AGCGGCCGGG CMTAMGM 420
GATCCCGCCC TGTTGATGGG
AGAACCATTT TCCTMTTTT AGCTGGTCGC GCATA ATG 474
CAMTTATTG GAT GAT
1 Met Asp Asp
5
1
CAG CAA GCT TTG ATGCM GAT TTG GCT GTC 522
MT TCA ATC CTT CAT MG
Gln Gln Ala Leu MetGln Asp Leu Ala Val
Asn Ser Ile Leu His Lys
5 10 15
2 CCA GTC GGC CAG TACMG MA CCA GGA MG CM 570
O CAT TAT CTT MC CTT
Pro Val Gly Gln TyrLys Lys Pro Gty Lys
His Tyr Leu Gln Asn Leu
20 25 30 35
CAT CAC CM MA MC ATGTTC GAG TCA MT TTG 618
AGA ATG MC ATA GAG
His His Gln Lys MetPhe Glu Ser Asn Leu
Asn Arg Net Asn lle Glu
2 40 45 50
5
GAG GM MA AGG ATC GTTACT AGA CCA GTT MA 666
CTG CAG CTA GM GAC
Glu Glu Lys Arg ValThr Arg Pro Val Lys
Ile Leu Gln Leu Glu Asp
55 60 65
CTG AGA TCT MG TCT GCCTTT GGG CAG TCT ATG 714
MG ATC GAT CTA CAC
3 Leu Arg Ser Lys AlaPhe Gly Gln Ser llet
0 Ser Lys Ile Asp Leu His
70 7580
TAT ACC MC MT GAG ATTCCG TTA ACT ACC CAA 762
TTG GTA GAT GAC TTG
Tyr Thr Asn Asn IlePro Leu Thr Thr Gln
Glu Leu Val Asp Asp Leu
85 90 95
3 GAC AM GCT GTG GM GATCGC AGT ATT CAC ATG 810
5 CTG CTG MG AGT CTC
Asp Lys Ala Val AspArg Ser Ile His Ilet
Glu Leu Leu Lys Ser Leu
100 105 110 115
MG ATA TTA CTT GTA GGGAGT ACA CAG GCT ACT 858
GTA MT MT TTA GM
Lys Ile Leu Leu GlySer Thr Gln Ala Thr
Val Vat Asn Asn Leu Glu
4 120 125 130
0
CCA TCA CCG TCA TTGMT MT ACA CCA CTT 906
CCA GM GAT GGT GCA GAG
Pro Ser Pro Ser LeuAsn Asn Thr Pro Leu
Pro Glu Asp Gly Ala Glu
135 140 145
AGG AM MG CGG CTA GTAGGT CCC CCT MT AGG 954
TCT GTA GAT AGA AGT
4 Arg Lys Lys Arg ValGly Pro Pro Asn Arg
5 Leu Ser Val Asp Arg Ser
150 155160
TCC CCT CCT CCA CCAGAC ATA CTA CAC CAG 1002
GGA TAC ATT ATT GCC CGG
Ser Pro Pro Pro ProAsp Ile Leu His Gln
Gly Tyr Ile Ile Ala Arg
165 170 175
21 ~3052~
WO 95/28421 PCT/US94/11690
-30-
MT GGG TCA TTC ACT AGC ATC MC AGT GAA GGA 1050
GAG TTC ATT CCA GAG
Asn Gly Ser Phe Thr Ser Ile Asn Ser Glu
Gly Glu Phe Ile Pro Glu
180 185 190 195
AGC ATG GAC CM ATG CTG GAT CCA TTG TCT 1098
TTA AGC AGC CCT GM MT
Ser Met Asp Gln lief Leu Asp Pro Leu Ser
Leu Ser Ser Pro Glu Asn
200 205 210
TCT GGC TCA GGA AGC TGT CCG TCA CTT GAT 1146
AGT CCT TTG GAT GGA GAA
Ser Gly Ser Gly Ser Cys Pro Ser Leu Asp
Ser Pro Leu Asp Gly Gtu
215 220 225
1 AGC TAC CCA AM TCA CGG ATG CCT AGG GCA 1194
O CAG AGC TAC CCA GAT MT
Ser Tyr Pro Lys Ser Arg lief Pro Arg Ala
Gln Ser Tyr Pro Asp Asn
230 235 240
CAT CAG GAG TTT ACA GAC TAT GAT MC CCC 1242
ATT TTT GAG MA TTT GGA
His Gln Glu Phe Thr Asp Tyr Asp Asn Pro
Ile Phe Glu Lys Phe Gly
245 250 255
AM GGA GGA ACA TAT CCA AGA AGG TAC CAC 1290
GTT TCC TAT CAT CAC CAG
Lys Gly Gly Thr Tyr Pro Arg Arg Tyr His
Val Ser Tyr His Nis Gln
260 265 270 275
GAG TAT MT GAC GGT CGG MG ACT TTT CCA AGA 1338
GCT AGA AGG ACC CAG
2 Glu Tyr Asn Asp Gly Arg Lys Thr Phe Pro
0 Arg Ala Arg Arg Thr Gln
280 285 290
GGC ACC AGT TTC CGG TCT CCT GTG AGC TTC 1386
AGT CCT ACT GAT CAC TCC
Gly Thr Ser Phe Arg Ser Pro Val Ser Phe
Ser Pro Thr Asp His Ser
295 300 305
2 TTA AGC ACT AGT AGT GGA AGC AGT GTC TTT 1434
5 ACC CCA GAG TAT GAC GAC
Leu Ser Thr Ser Ser Gly Ser Ser Val Phe
Thr Pro Glu Tyr Asp Asp
310 315 320
AGT CGA ATA AGA AGA CGG GGG AGT GAC ATA 1482
GAC MT CCT ACT TTG ACT
Ser Arg Ile Arg Arg Arg Gly Ser Asp Ile
Asp Asn Pro Thr Leu Thr
3 325 330 335
0
GTC ACA GAC ATC AGC CCA CCC AGC CGT TCA 1530
CCT CGA GCT CCG ACC MC
Val Thr Asp Ile Ser Pro Pro Ser Arg Ser
Pro Arg Ala Pro Thr Asn
340 345 350 355
TGG AGA CTG GGC MG CTG CTT GGC CM GGA GCT 1578
TTT GGT AGG GTC TAC
3 Trp Arg Leu Gly Lys Leu Leu Gly Gln Gly
5 Ala Phe Gly Arg Val Tyr
360 365 370
CTC TGC TAT GAT GTT GAT ACC GGA AGA G11G 1626
CTG GCT GTT MG CAA GTT
Leu Cys Tyr Asp Yal Asp Thr Gly Arg Glu
Leu Ala Yal Lys Gln Val
375 380 385
4 CAG TTT MC CCT GAG AGC CCA GAG ACC AGC 1674
O MG GM GTA MT GCA CTT
Gln Phe Asn Pro Glu Ser Pro Glu Thr Ser
Lys Glu Val Asn Ala Leu
390 395 400
GAG TGT GM ATT CAG TTG TTG AM MC TTG TTG 1722
CAT GAG CGA ATT GTT
Glu Cys Glu Ile Gln Leu Leu Lys Asn Leu
Leu His Glu Arg Ile Val
4 405 410 415
5
CAG TAT TAT GGC TGT TTG AGG GAT CCT CAG 1770
GAG MA ACA CTT TCC ATC
Gtn Tyr Tyr Gly Cys Leu Arg Asp Pro Gln
Glu Lys Thr Leu Ser Ile
420 425 430 435
TTT ATG GAG CTC TCG CCA GGG GGT TCA ATT 1818
MG GAC CAA CTA AM GCC
5 Phe lief Glu Leu Ser Pro Gly Gly Ser Ile
0 lys Asp Gln Leu Lys Ala
440 445 450
TAC GGA GCT CTT ACT GAG MC GTG ACG AGG 1866
MG TAC ACC CGT CAG ATT
Tyr Gly Ala Leu Thr Glu Asn Val Thr Arg
Lys Tyr Thr Arg Gln Ile
455 460 465
_ WO 95/28421 218 6 5 ~ 6 PCT/US94/11690
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CTG GGGGTC TATTTG CAT ATGATT AG11 1914
GAG CAT ACT MT GTC GAT
CAT
Leu GlyVatNisTyrLeu His Net1le HisArg
Glu Ser Asn Yal Asp
470 475 480
ATC GGAGCAMT ATCTTA AGG ACAGGC ATCMG TTA 1962
MA GAT TCC MT
Ile GlyAlaAsnIleLeu Arg ThrGly IleLys
Lys Asp Ser Asn Leu
485 490 495
GGA TTTGGGGCTAGTAM CGG ACCATC CTCTCA 2010
GAC CTT CAG TGT GGC
Gly PheGlyAlaSerLys Arg ThrIle LeuSer
Asp Leu Gln Cys Gly
500 505 510 515
1 ACA ATGMGTCTGTCACA GGC TACTGG AGTCCT 2058
O GGA ACG CCA ATG GAG
Thr lletLysSerValThr Gly TyrTrp SerPro
Gly Thr Pro /let Glu
520 525 530
GTC AGTGGAGM GGCTAT GGA GCAGAC TGGAGT 2106
ATC AGA AAA ATC GTA
Vat SerGlyGluGlyTyr Gly AlaAsp TrpSer
Ile Arg Lys Ile Val
535 540 545
GCA ACTGTGGTAGMATG CTA MGCCA TGGGCT 2154
TGT ACT GAA CCT GAA
Ala ThrVelValGlu/let Leu LysPro TrpAls
Cys Thr Glu Pro Glu
550 555 560
TTT GCAATGGCTGCCATC TTT GCCACT CCAACG 2202
GM MG ATC CAG MC
2 Phe AlafiletAlaAlaIle Phe AlaThr ProThr
0 Glu Lys Ile Gln Asn
565 570 475
CCA CTGCCACCTCATGTC TCA ACTCGG TTCCTC 2250
MG GAC TAT GAC AM
Pro LeuProProHisVal Ser ThrArg PheLeu
Lys Aap Tyr Asp Lys
580 585 590 595
2 CGG TTTGTAGAGGCCAM CTT TCAGCG GJ1GCTC 2298
5 ATT CGA CCT GAG TTG
Arg PheValGluAlaLys Leu SerAle GluLeu
Ile Arg Pro Glu Leu
600 605 610
CGG ATGTTTGTGCATTAT CAC 2352
CAC TAGCAGCGGC
GGCTTCGGTC
CTCCACCAGC
Arg MetPheValNisTyr Nis
Nis
3 615 620
0
TCCATCCTCG TTTTTATAM 2412
CGGCCACCTT AMGAGAGAT
CTCTCTTACT
GCACTTTCCT
GGGGAGAAM TTGGTTAMT 2472
AGACMGAGG TTGTTTMTA
GAAMTATTT
CTCTTGATTC
ATMTAGTM 2503
ACTAAAAAM
AAAAAAAAM
A
An MEKK protein of the present invention, referred to
35 here as MEKK 3, includes an MEKK protein having at least a
portion of the nucleic acid and/or amino acid sequence
shown in Table 3 and represented by SEQ ID N0:5 and SEQ ID
N0:6, respectively.
Table 3.
4 O AGGGMCAM CCACCGCGGTGGCGGCCGCTCTAGMCTAGTGGATCCCCC60
AGCTGGAGCT
GGGCTGCAGG MTTCGGCACGAGGMCAGTGGCCGGTCGGAGCGTCTTCTGGACTTCAGG120
ACTCGCAGGC GGCCCGGTCGAGTGGCGCCGCCGAGGCCGGGTTGGGCCG11GCCTGGGAGC180
GCCGGGGATG TAGCGGGCCAACCTGCTCATGCCACAGCGCCCGGCCGCGGCCGAGCCGGA240
GCCTGGGGAG GCGGCGGGGGCCCCGAGCGCAGCCCACGGCCCCCGCGCGGAGCCAGGCCC300
4 5 GCTGCCGTCCCCGCCGCCCGCTCCCCCGGCATGCAGCCCCGGCTGCGGAGGTGACACTTC360
TGGGCTGTAG TCGCCACCGCCGCCTCCGCCATCGCCACC CM GAG 414
ATG
GAT
GAA
/let Gln
Asp Glu
Glu
1 5
21 ~b52~
WO 95/28421 PCTlUS94/11690
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GCA TTA TCGATCATG CTG GCCCTC ATGAGCCGA 462
GAC MG GTG CAG
GAC
Ala Leu SerIleHet AspLeu AlaLeu MetSerArg
Asp Lys Val Gln
10 15 20
CGA ACC TTGTCTGGA GAGACC MGMT GACACAGGT 510
CGG TAT ATG MG
Arg Thr LeuSerGly GluThr LysAsn AspThrGly
Arg Tyr llet Lys
25 30 35
CAC CG AGGCAGAGT GTCAGA MGTTT CACMTGGG 558
MC GAC ATC GM
His Pro ArgGlnSer VatArg LysPhe HisAsnGly
Asn Asp Ile Glu
40 45 50
1 GAG AGA ATTATAGCA AGCCGG GTGAGA GAAGATGTG 606
O CGA TTC CCT TAC
Glu Arg ileIleAla SerArg ValArg GluAspVal
Arg Phe Pro Tyr
55 60 65
GAG CAC GTGACAACA TTTGGG CCTCTT TTGCATTAT 654
MG GTC CAG GAT
Glu His ValThrThr PheGly ProLeu LeuHisTyr
Lys Val Gln Asp
70 75 80 85
ATG MT GAGCTCTCC CTGTTG MCCM GATCTCGAT 702
MT ATC AM GAT
lletAsn GluLeuSer LeuLeu AsnGln AspLeuAsp
Asn lle Lys Asp
90 95 100
AM GCC GACATTTTG AGAAGC AGTATG AGCCTTAGG 750
ATT GAT TCA MA
2 Lys Ala AspIleLeu ArgSer Serllet SerLeuArg
0 Ile Asp Ser Lys
105 110 115
ATA CTA TTATCCCM AGAMC ACTAGT TCTCCCCAC 798
CTG GAC CAT TCC
Ile Leu LeuSerGln ArgAsn ThrSer SerProHis
Leu Asp His Ser
120 125 130
2 TCT GG11 TCCAGGCAG CGGATC CCTTCC TCTGCAGGG 846
5 GTG GTT MG CAG
Ser Gly SerArgGln ArgIle ProSer SerAlsGly
Val Val Lys Gln
135 140 145
GAT ATA ACCATCTAC GCTCCT CCCAGA AGGCACCTG 894
MT CM GAG AGC
Asp Ile ThrIleTyr AlaPro ProArg ArgHisLeu
Asn Gln Glu Ser
3 150 155 160 165
0
TCT GTC TCCCAGMC GGCCGA TCTCCT CCGGGATAT 942
AGC CCT AGC CCC
Ser Yal SerGlnAsn GlyArg SerPro ProGlyTyr
Ser Pro Ser Pro
170 175 180
GTA CCT CGACM CAG ATTGCC CMGGA TATACGAGC 990
GAG CAC CGG TCC
3 Val Pro ArgGlnGln IleAla GlnGly TyrThrSer
5 Glu His Arg Ser
185 190 195
ATC MC GAAGGTGAA ATCCCA ACCAGC CAGTGTATG 1038
AGC TTC GAG GAA
Ile Asn GluGlyGlu IlePro ThrSer GlnCysIlet
Ser Phe Glu Glu
200 205 210
4 CTA GAT CTCAGCAGT GAAMT TTGTCA AGCTGCCM 1086
O CCC GCC TCC GGA
Leu Asp LeuSerSer GluAsn LeuSer SerCysGln
Pro Ala Ser Gly
215 220 225
TCC TTG AGGTCAGCA AGCCCA TTCAGG TCACMATG 1134
GAC GAC TCC AAA
Ser Leu ArgSerAla SerPro PheArg SerGlnIlet
Asp Asp Ser Lys
4 230 235 240 245
5
TCC CGA CGGAGCTTC GACMC MGGM TCAGATCGG 1182
GCC CCA AGA TGC
Ser Arg ArgSerPhe AspAsn LysGlu SerAspArg
Ala Pro Arg Cys
250 255 260
GAG ACC CTCTATGAT GGTGTC GGTGGA TATCCCAGG 1230
CAG MA MA ACC
5 Glu Thr LeuTyrAsp GlyVal GlyGly TyrProArg
0 Gln Lys Lys Thr
265 270 275
CGC TAC GTGTCTGTG CACMA TACMT GGCAGAAGA 1278
CAT CAT GAC GAT
Arg Tyr ValSerVal HisLys TyrAsn GlyArgArg
His His Asp Asp
280 285 290
WO 95/28421 2.18 5 5 ~ ~ pCT/US94/11690
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ACA TTT CCC CG11 ATA CG11 CGG CAT CM GGC 1326
MC CTA TTC ACT CTG GTG
Thr Phe Pro Arg Ile Arg Arg His Gln Gly
Asn Leu Phe Thr Leu Yal
295 300 305
CCC TCA AGT CGC TCC TTG AGC ACA MT GGC 1374
GAG MC ATG GGT GTA GCT
Pro Ser Ser Arg Ser Leu Ser Thr Asn Gly
Glu Asn Met Gly Val Ala
310 315 320 325
GTG CM TAC CTG GAC CCC CGT GGG CGC CTA 1422
CGG AGT GCA GAC AGT GAG
Vat Gln Tyr Leu Asp Pro Arg Gly Arg Leu
Arg Ser Ala Asp Ser Glu
330 335 340
1 MT GCC CTC ACT GTG CAG GM AGG MT GTG CCA 1470
O ACC MA TCT CCT AGT
Asn Ala Leu Thr Val Gln Glu Arg Asn Val
Pro Thr Lys Ser Pro Ser
345 350 355
GCT CCC ATC MT TGG CGT CGG GGG MG CTC 1518
CTG GGT CM GGT GCC TTC
Ala Pro Ile Asn Trp Arg Arg Gly Lys Leu
Leu Gly Gln Gly Ala Phe
360 365 370
GGC AGG GTC TAC TTG TGC TAT GAT GTG GAC 1566
ACA GGA CGT GAA CTT GCT
Gly Arg Vat Tyr Leu Cys Tyr Asp Val Asp
Thr Gly Arg Glu Leu Ala
375 380 385
TCT MG CAG GTC CAG TTT GAC CCA GAT AGT 1614
CCT GAG ACA AGC MG GAG
2 Ser Lys Gln Val Gln Phe Asp Pro Asp Ser
0 Pro Glu Thr Ser Lys Glu
390 395 400 405
GTG AGT GCT CTG GAG TGT GAG ATC CAG TTG 1662
CTG MG MC CTG CAG CAT
Val Ser Ala Leu Glu Cys Glu Ile Gln Leu
Leu Lys Asn Leu Gln His
410 415 420
2 GAG CGC ATT GTG CAG TAC TAC GGC TGC CTG 1710
5 CGG GAC CGT GCT GAG MG
Glu Arg Ile Vat Gln Tyr Tyr Gly Cys Leu
Arg Asp Arg Als Glu Lys
425 430 435
ATC CTC ACC ATC TTT ATG GAG TAT ATG CCA 1758
GGG GGC TCT GTA MA GAC
Ile Leu Thr Ile Phe Met Glu Tyr Met Pro
Gly Gly Ser Val Lys Asp
3 440 445 450
0
CAG TTG MG GCC TAC GGA GCT CTG ACA GAG 1806
AGT GTG ACC CGC MG TAC
Gln Leu Lys Ala Tyr Gly Ala Leu Thr Glu
Ser Val Thr Arg Lys Tyr
455 460 465
ACC CGG CAG ATT CTG GAG GGC ATG TCA TAC 1854
CTG CAC AGC MC ATG ATT
3 Thr Arg Gln Ile Leu Glu Gly Met Ser Tyr
5 Leu His Ser Asn Met lle
470 475 480 485
GTG CAT CGG GAC ATC MG GGA GCC MT ATC 1902
CTC CGA GAC TCA GCT GGG
Val His Arg Asp Ile Lys Gly Ala Asn Ile
Leu Arg Asp Ser Ala Gly
490 495 500
4 MT GTG MG CTT GGG GAT TTT GGG GCC AGC 1950
O MA CGC CTA CAG ACC ATC
Asn Val Lys Leu Gly Asp Phe Gly Ala Ser
Lys Arg leu Gln Thr Ile
505 510 515
TGC ATG TCA GGG ACA GGC ATT CGC TCT GTC 1998
ACT GGC ACA CCC TAC TGG
Cys Met Ser Gly Thr Gly Ile Arg Ser Val
Thr Gly Thr Pro Tyr Trp
4 520 525 530
5
ATG AGT CCT GAA GTC ATC AGT GGC GAG GGC 2046
TAT GGA AGA MG GCA GAC
Met Ser Pro Glu Val Ile Ser Gly Glu Gly
Tyr Gly Arg Lys Ala Asp
535 540 545
GTG TGG AGC CTG GGC TGT ACT GTG GTG GM 2094
ATG CTG ACA GAG AM CCA
5 Val Trp Ser Leu Gly Cys Thr Val Val Glu
0 Met Leu Thr Glu Lys Pro
550 555 560 565
CCT TGG GCA GAG TAT GM GCT ATG GCT GCC 2142
ATT TTC MG ATT GCC ACC
Pro Trp Ala Glu Tyr Glu Ala Met Ala Ala
Ile Phe Lys Ile Ala Thr
570 575 580
z~855z6
WO 95/28421 PCT/US94111690
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CAG CCT ACC MT CCT CAG CTG TCA GM CAC 2190
CCC TCT CAC ATC GGC AGG
Gln Pro Thr Asn Pro Gln Leu Ser Glu His
Pro Ser His Ile Gly Arg
585 590 595
GAC TTC CTG AGG CGC ATA TTT CAG AGA CCC 2238
GTG GM GCT CGT TCA GCT
Asp Phe Leu Arg Arg Ile Phe Gln Arg Pro
Yal Glu Ala Arg Ser Ala
600 605 610
GAG GAG CTG CTC ACA CAC CAC GTG TAC TGAGCTCTCA2287
TTT GCA CAG CTA
Glu Glu Leu Leu Thr His His Val Tyr
Phe Ala Gln Leu
615 620 625
1 AGGCTATCAG GCTGCCAGCT GCCACCTGCTGGGGCTGCTG 2347
O GAGCAGGCM TCAGGCTCAG
TGMGTTGCT GCTTCTTCCA GGCMGGCTAAGCATCGGTC 2407
TGACCAGTGG CAGCCATTGT
TTGTCTGTGC CCCATCTGCC ACTGGGACTCTGGGATAGCT 2467
AMGCCAGGA CTGGCATCM
GACTGGGAGC TCCAGCCTGT MGACCCMGCCTTMGCTC AGTATGGCGG2527
AGCTTTAGCA
GMGGGCTGG AMCAGTATG CMGACTGCCCCTACCCTCA 2587
ATGGGTCCTG GATGTGTCCT
Z MCACTGCAG ACAGCACTGA AGTCMGAGGCAGGAGGTCC 2647
5 GACTGGGGCA TCMGGGTAT
GMTAGTGTT ACTTCATTCA GAGTGTTACTCCCMTGTTT GGAGACCACC2707
TTGTTTCTCT
AGCCTGTCTC TGGGCTGCM GCCTGAGGTATCCCCCAGCC 2767
MGCCCAGCA MCAGMGGT
AGAGGTTTGG GCTACCCCAC TATAGCTTCCTGTCAGTCCT 2827
AGGTATTCGG GTCTTACCM
AGATGMTGA AGCAMTGTT ACACTGCCTTGGAGGAGCTA 2887
ATTCTGGGM CTCGGATMG
2 CAGGGCCTGA GAGATGGAGC TGCCTCCAGAACCCAGTCTT 2947
O MCTGGGGAG GTCMTGCM
TTGTCTCTGT TTTACMGTT GGAGTCACTCCCCAGTTTTA 3007
TTATGCTGTT AMCTGGAGA
CTTTGCCCTC TGAGCTCTGG AGACCCATGTTTGGACTGGA 3067
GGGCTTAGGC TGGMGAGCT
GATGGCCTCT GCCCCTGGCC TG 3089
An MEKK protein of the present invention can also
25 include an MEKK protein having at least a portion of the
nucleic acid and/or amino acid sequence shown in Table 4
and represented by SEQ ID N0:7 and SEQ ID N0:8,
respectively, and is referred to as MEKK 4.
Table 4.
3 MTTCGGCAC 60
O GAGMCCTAT
CAGACATTGG
CTGGCCAGTG
TTTGAA11TCC
CCTCCCCTCG
GCCGTCCMGGGCTACGAGCCAGAGGACGA GGTCGAGGAC ACGGAGGTTG120
AGCTGAGGGA
GCTGGAGAGCGGGACGGAGGAGAGTGACGA GGAGCCMCC CCCAGTCCGA180
GGGTGCCAGA
GCTCAGGCTGTCCACAGACACCATCTTGGA CAGTCGCTCC CAGGGCTGCG240
TCTCCAGGM
GCTGGAGAGGCTCGAGTCAGAGGMGATTC CATAGGCTGG GGGACAGCGG300
ACTGTGGCCC
3 TGMGCCAGCAGGCATTGTTTGACTTCTAT CTATAGACCA TTCGTGGACA360
5 MGCACTGM
GCAAATGGGGCTMGAAAGTTMTTTTACG ACTTCATMG CTTATGMTG420
GGTCCTTGCA
MGAGCTCGTGTAGCTCTGGTGAAGGACGA CCGTCAGTGG AGTTCTCTGA480
CTTTCCAGGT
CCCATGTGGGGCTCGGATTATGTGCAGTTG TCGGGMCAC CTCCTTCCTC540
AGAGCAGAI1G
TGTAGCGCTGTGTCCTGGGAAGMCTGAGA GCCATGGACC TGCCTTCCTT600
TGAGCCCGCC
4 TTCCTGGTGCTCTGTCGGGTCCTGCTGMC GTGATCCACG AGTGCCTGAA660
O GCTGCGGCTG
GMCAGAGGCTGCCGGGGAGCCTTCCCTCT TGAGTATCAA ACAGCTAGTG720
CGAGAGTGTA
MGAGGTCCTAMGGGCGGGCTCCTG ATG MG CAG TAT TAC 7T3
CAG TTC ATG CTG
lief Lys Gln Tyr Tyr Gln
Phe Met Leu
1 5
4 5 CAG GAG GTC CTG GGC GGA CTG GAG MG ACC GAC TGC MC ATG GAT GCC 821
Gln Glu Val Leu Gly Gly Leu Glu Lys Thr Asp Cys Asn lief Asp Ala
15 20 25
TTT GAG GAG GAC CTG CAG MG ATG CTG ATG GTG TAT TTT GAT TAC ATG 869
Phe Glu Glu Asp Leu Gln Lys lief Leu lief Val Tyr Phe Asp Tyr Met
5 0 30 35 40
AGA AGC TGG ATC CM ATG CTA CAG CAG TTA CCT CAG GCT TCC CAT AGC 917
Arg Ser Trp lie Gln Met Leu Gln Gln Leu Pro Gln Ala Ser His Ser
45 50 55
21 ~3~526
WO 95/28421 PCT/US94/111i90
-35-
TTA AM MC CTG CTA GM GAG GM TGG MT TTC 965
ACC AM GM ATA ACC
Leu Lys Asn Leu Leu Glu Glu Glu Trp Asn
Phe Thr Lys Glu lle Thr
60 65 70
GT TAT ATC CGT GGC GGA GM GCG GG GCT GGA 1013
MG CTT TTC TGT GAC
His Tyr Ile Arg Gly Gly Glu Ala Gln Ala
Gly Lys Leu Phe Cys Asp
75 80 95
ATC GG GGG ATG CTG CTG AM TCC AG GGG AGC 1061
TTT CTG GAA TCC GGC
Ile Ata Gly Met Leu Leu Lys Ser Thr Gly
Ser Phe Leu Glu Ser Gly
90 95 100 105
1 CTG GG GAG AGC TGT GCT GAG CTG TGG ACC 1109
O AGN GCC GAC GAC MC GGT
Leu Gln Glu Ser Cys Ala Glu Leu Trp Thr
Xaa Ala Asp Asp Asn Gly
110 115 120
GCT GCC GAC GAG CTA AGG AGA TCT GTC ATC 1157
GAG ATC AGC CGA GG CTC
Ala Ala Asp Glu Leu Arg Arg Ser Val Ile
Glu Ile Ser Arg Ala Leu
125 130 135
MG GAG CTC TTC CAC GM GCC AGG GM AGA GCC 1205
TCC MG GCC CTG GGC
Lys Glu Leu Phe His Glu Ala Arg Glu Arg
Ala Ser Lys Ala Leu Gly
140 145 150
TTT GCT AM ATG CTG AGG MG GAC CTA GM ATA 1253
GG GG GAG TTC GTG
2 Phe Ala Lys Net Leu Arg Lys Asp Leu Glu
0 Ile Ala Ala Glu Phe Val
155 160 165
CTA TCT GG TG GCC CGA GAG CTC CTG GAC 1301
GCT CTG AM GG MG GG
Leu Ser Ala Ser Ala Arg Glu Leu Leu Asp
Ala Leu Lys Ala Lys Gln
170 175 180 185
2 TAT GTT MG GTA GG ATT CCC GGG TTA GAG 1349
5 MT TTG GC GTG TTT GTC
Tyr Val Lys Val Gln Ile Pro Gly Leu Glu
Asn Leu His Val Phe Val
190 195 200
CCC GAC AGC CTC GCT GAG GAG MG MA ATT 1397
ATT TTG GG CTA CTC MT
Pro Asp Ser Leu Ala Glu Glu Lys Lys Ile
Ile Leu Gln Leu Leu Asn
3 205 210 215
0
GCT GCC AG GGA MG GAC TGC TG MG GAT CG 1445
GAC GAC GTC TTC ATG
Ala Ala Thr Gly Lys Asp Cys Ser Lys Asp
Pro Asp Asp Val Phe Ilet
220 225 230
GAT GCC TTC CTG CTC CTG ACC MG GT GGG 1493
GAC CGA GCC CGT GAC TG
3 Asp Ala Phe Leu Leu Leu Thr Lys His Gly
5 Asp Arg Als Arg Asp Ser
235 240 245
GM GAT GGC TGG GGC AG TGG GAA GCT CGG 1541
GCT GTC AAA ATT GTG CCT
Glu Asp Gly Trp Gly Thr Trp Glu Ala Arg
Ala Val Lys Ile Val Pro
250 255 260 265
4 CAG GTG GAG ACT GTG GAC ACC CTG AGA AGC 1589
O ATG GG GTG GAC MC CTT
Gln Val Glu Thr Val Asp Thr Leu Arg Ser
Ret Gln Yal Asp Asn Leu
270 275 280
CTG CTG GTT GTC ATG GAG TCT GCT GC CTC 1637
GTA CTT GG AGA AM GCC
Leu Leu Val Val llet Glu Ser Ala His Leu
Val Leu Gln Arg Lys Ale
4 285 290 295
5
TTC GG CAG TCC ATT GAG GGG CTG ATG ACT 1685
GTA CGC GT GAG GG AG
Phe Gln Gln Ser Ile Glu Gly Leu llet Thr
Val Arg His Glu Gln Thr
300 305 310
TCT AGC GG CCC ATC ATC GCC AM GGT TTG 1733
GG GG CTC MG MC GAT
5 Ser Ser Gln Pro Ile Ile Ala Lys Gly Leu
0 Gln Gtn Leu Lys Asn Asp
315 320 325
GCA CTT GAG CTA TGC MC AGA ATC AGC GAT 1781
GCC ATC GAC CGT GTG GAC
Ala Leu Glu Leu Cys Asn Arg Ile Ser Asp
Ala Ile Asp Arg Val Asp
330 335 340 345
2~~b5?6
WO 95/28421 PCT/US94/11690
-36-
GC ATG TTC ACC CTG GAG TTC GAT GCT GAG 1829
GTC GAG GJ1G TCT GAG TCG
His llet Phe Thr Leu Glu Phe Asp Ala Glu
Val Glu Glu Ser Glu Ser
350 355 360
GCC ACG CTG GG GG TAC TAC CGA GM GCC ATG 1877
ATT GG GGC TAC MC
Ala Thr Leu Gln Gln Tyr Tyr Arg Glu Ala
llet Ile Gln Gly Tyr Asn
365 370 375
TTT GGG TTT GAG TAT GT AM GAA GTT GTT 1925
CGT TTG ATG TCT GGG GAA
Phe Gly Phe Glu Tyr Nis Lys Glu Val Val
Arg Leu Het Ser Gly Glu
380 385 390
ZO TTC AGG GG MG ATA GGA GAC MA TAT ATA ACG 1973
TTC GCC GG MG TGG
Phe Arg Gln Lys Ile Gly Asp Lys Tyr Ile
Thr Phe Ala Gln Lys Trp
395 400 405
ATG MT TAC GTG CTG ACC AAA TGC GAG AGC 2021
GGC AGA GGC AG AG11 CCC
het Asn Tyr Val Leu Thr Lys Cys Glu Ser
Gly Arg Gly Thr Arg Pro
1 410 415 420 425
5
AGA TGG GCC ACC CM GGA TTT GAT TTC CTA 2069
CAA GCC ATT GAA CCT GCC
Arg Trp Ala Thr Gln Gly Phe Asp Phe Leu
Gln Ala lle Glu Pro Ala
430 435 440
TTT ATT TG GCT TTA CG GAIL GAT GAC TTC 2117
TTG AGT TTG CM GCC CTG
2 Phe lle Ser Ala Leu Pro Glu Asp Asp Phe
0 Leu Ser Leu Gln Ala Leu
445 450 455
ATG MT GAG TGC ATC GGG GC GTC ATA GGA 2165
MG CG GC AGC CCT GTC
Net Asn Glu Cys Ile Gly His Val Ile Gly
Lys Pro His Ser Pro Val
460 465 470
2 AG GCT ATC GT CGG MC AGC CCC CGC CCT GTG 2213
5 MG GTG CCC CGA TGC
Thr Ala Ile His Arg Asn Ser Pro Arg Pro
Val Lys Val Pro Arg Cys
475 480 485
GC AGT GAC CCT CCT MC CCT GC CTC ATC ATC 2261
CCG ACT CG GAG GGA
Nis Ser Asp Pro Pro Asn Pro Nis Leu lle
Ile Pro Thr Pro Glu Gly
3 490 495 500 505
0
TTC AGG GGT TCC AGT GTC CCT GAA MC GAC 2309
CGC TTG GCC TCC ATA GCT
Phe Arg Gly Ser Ser Yal Pro Glu Asn Asp
Arg Leu Ala Ser Ile Ala
510 515 520
GG GM CTG GG TTC AGG TCT CTG AGT CGG GC 2357
TG AGC CCC ACG GAA
3 Ala Glu Leu Gln Phe Arg Ser Leu Ser Arg
5 His Ser Ser Pro Thr Glu
525 530 535
GAG CGA GAC GAG CG GCG TAT CCT CGG AGT 2405
G11C TG AGT GGA TG ACT
Glu Arg Asp Glu Pro Ala Tyr Pro Arg Ser
Asp Ser Ser Gly Ser Thr
540 545 550
4 CGG AGA AGC TGG GM CTT CGA AG CTC ATC 2453
O AGC GG ACC AM GAC TCG
Arg Arg Ser Trp Glu Leu Arg Thr Leu Ile
Ser Gln Thr Lys Asp Ser
555 560 565
GCC TCT MG GG GGG CCC ATA GM GCT ATC GG 2501
MG TG GTC CGA CTG
Ala Ser Lys Gln Gly Pro Ile Glu Ala Ile
Gln Lys Ser Val Arg Leu
4 570 575 580 585
5
TTT GM GAG AGG AGG TAT CGA GAG ATG AGG AGA MG MT ATC ATC GGC 2549
Phe Glu Glu Arg Arg Tyr Arg Glu llet Arg Arg Lys Asn Ile Ile Gly
590 595 600
GA GTG TGC GAT ACC CCT MG TCC TAT GAT MC GTC ATG GT GTT GGA 2597
5 0 Gln Val Cys Asp Thr Pro Lys Ser Tyr Asp Asn Val llet Nis Yal Gly
605 610 615
CTG AGG MG GTG ACA TTT MG TGG CM AGA GGA MC AM ATT GGA GM 2645
Leu Arg Lys Val Thr Phe Lys Trp Gln Arg Gly Asn Lys Ile Gly Glu
620 625 630
216526
WO 95/28421 PCT/US94111690
-37-
GGA CAG TAT GG11 MA GTA TAC ACC TGC ATC 2693
AGT GTT GAC ACA GGG GAG
Gly Gln Tyr Gly Lys Val Tyr Thr Cys Ile
Ser Val Asp Thr Gly Glu
635 640 645
CTG ATG GCC ATG MG GAG ATT CGA TTT CAG 2741
CCT MC GAC CAC MG ACT
Leu Ret Ala lief Lys Glu 1le Arg Phe Gln
Pro Asn Asp Nis Lys Thr
650 655 660 665
ATC MG GAG ACT GCA GAC GAG TTG MA ATA TTT 2789
GM GGC ATC MG CAC
Ile Lys Glu Thr Ala Asp Glu Leu Lys Ile
Phe Glu Gly 1le Lys Nis
670 675 680
1 CCC MC CTG GTC CGG TAT TTT GGC GTG GAG 2837
O CTT CAC AGG GAA GAG ATG
Pro Asn Leu Val Arg Tyr Phe Gly Val Glu
Leu His Arg Glu Glu Ret
685 690 695
TAC ATC TTC ATG GAG TAC TGT GAT GAG GGT 2885
ACA CTA GAG GAG GTG TCA
Tyr 1le Phe Met Glu Tyr Cys Asp Glu Gly
Thr Leu Glu Glu Val Ser
700 705 7t o
CGA CTG GGC CTG CAG GAG CAC GTC ATC AGG 2933
TTA TAT ACC MG CAG ATC
Arg Leu Gly Leu Gln Glu Nis Val lie Arg
Leu Tyr Thr Lys Gln lie
T15 720 725
ACT GTC GCC ATC MC GTC CTC CAT GAG CAC 2981
GGC ATC GTT CAC CGA GAC
2 Thr Val Ala 1le Asn Val Leu Nis Glu His
0 Gly Ile Yal His Arg Asp
730 735 ~ 740 745
ATC AM GGT GCC MT ATC TTC CTT ACG TCA TCT 3029
GGA CTA ATC MG CTG
Ile Lys Gly Ala Asn Ile Phe leu Thr Ser
Ser Gly Leu Ile Lys Leu
750 755 760
2 GG11 GAT TTT GGA TGC TCT GTA AM CTT MA 3077
5 MC MC GCC CAG ACC ATG
Gly Asp Phe Gly Cys Ser Yal Lys Leu Lys
Asn Asn Ala Gln Thr Ret
765 770 775
CCC GGA GAG GTG MC AGC ACC CTA GGG ACA 3125
GCA GCT TAC ATG GCC CCT
Pro Gly Glu Val Asn Ser Thr Leu Gly Thr
Ala Ala Tyr Met Ala Pro
3 780 785 790
0
GAJ1 GTT ATT ACC CGA GCC AM GGA GAA GGC 3173
CAC GGA CGT GCG GCA GAT
Glu Val Ile Thr Arg Ala Lys Gly Glu Gly
Nis Gly Arg Ala Ala Asp
795 800 805
ATC TGG AGT CTG GGG TGC GTC GTC ATA GAG 3221
ATG GTG ACT GGC MG CGG
3 Ile Trp Ser Leu Gly Cys Yal Val lie Glu
5 Ref Yal Thr Gly Lys Arg
810 815 820 825
CCT TGG CAT GAG TAT GM CAC MC TTT CAG ATT 3269
ATG TAC MG GTG GGG
Pro Trp His Glu Tyr Glu Nis Asn Phe Gln
Ile Ret Tyr Lys Yal Gly
830 835 840
4 ATG GGA CAC MG CCA CCA ATC CCG GM AGG CTA 3317
O AGC CCT GM GGA MG
Ret Gly His Lys Pro Pro Ile Pro Glu Arg
Leu Ser Pro Glu Gly Lys
845 850 855
GCC TTT CTC TCG CAC TGC CTG GM AGT GAC 3365
CCG MG ATA CGG TGG ACA
Ala Phe Leu Ser His Cys Leu Glu Ser Asp
Pro Lys Ile Arg Trp Thr
4 860 865 870
5
GCC AGC CAG CTC CTC GAC CAC GCT TTT GTC 3413
MG GTT TGC ACA GAT GM
Ala Ser Gln Leu Leu Asp Nis Ala Phe Vat
Lys Val Cys Thr Asp Glu
875 880 885
GAG T GMGTGMCC AGTCCGTGGC CTAGTAGTGT GTGGACAGAA3467
TCCCGTGATC
50 Glu
890
ACTACTGTAT GTMTATTTA CATAMGACT GCAGCGCAGG 3527
CGGCCTTCCT MCCTCCCAG
GACTGMGAC TACAGGGGTG ACMGCCTCA CTTCTGCTGC 3587
TCCTGTCGCC TGCTGAGTGA
CAGTGCTGAG GTTAMGGAG CCGCACGTTA AGTGCCATTA3647
CTACTGTACA CGGCCACCGC
5 CTCTGTCCCC TCCGACCCTC TCGTGACTGA GMCCMCCG 3T07
5 TGTCATCAGC ACAGTGTTTT
TGAGCTCCTG GGGTTCAGM GAACATGTAG TGTTCCCGGG TGTCCGGGAC GTTTATTTCA 3767
ACCTCCTGGT CGTTGGCTCT GACTGTGGAG CCTCCTTGTT CGAMGCTGC AGGTTTGTTA 3827
z ~ ~~5~s
WO 95/28421 PCT/US94/1169~
-38-
TGCAMGGCT CGTMGTGM GCTGAAGAM AGGTTCTTTT TCMTAMTG GTTTATTTTA 3887
GGAMGCGAA MMMAAM AAAAAA 3913
An MEKK protein of the present invention, referred to
here as MEKK 5, includes an MEKK protein having at least a
portion of the nucleic acid and/or amino acid sequence
shown in Table 5 and represented by SEQ ID N0:9 and SEQ ID
NO:lO, respectively.
Table 5.
MGMGMGG ACAGGGAGCA GAGGGGACM GAAMCACGG 60
CTGCTTTCTG GTTCMCCGA
1O TCGMCGMC TGATCTGGTT AGMCTGCAG GCCTGGCACG 120
CGGGCCGCAC CATCMTGAC
CAGGACCTCT TTCTCTACAC AGCCCGCCAG GCCATCCCAG 180
ACATCATCM TGAGATCCTC
ACCTTCAMG TTMCTACGG GAGCATTGCC TTCTCCAGCA 240
ATGGAGCCGG TTTCMCGGG
CCCTTGGTAG MGGCCAGTG CAGMCCCCT CAGGAGACM 300
ACCGTGTGGG CTGCTCATCG
TACCACGAGC ACCTCCAGCG CCAGAGGGTC TCGTTTGAGCATG 35T
AGGTGAAGCG GATA
tlet
1
GAG CTG CTG GAG TAC ATG GAG GCA CTT CTG 405
TAC CCA TCC TTG CAG GCT
Glu Leu Leu Glu Tyr llet Glu Ala Leu Leu
Tyr Pro Ser Leu Gln Ala
5 10 15
2 CAG MG GAC TAT GAA CGG TAC GCC GCC GTG 453
O MG GAC TTT GAG GAC AGA
Gln Lys Asp Tyr Glu Arg Tyr Ala Ala Val
Lys Asp Phe Glu Asp Arg
20 25 30
CAG GCG CTC TGC CTG TGG CTC MC ATC MG 501
ACG AM GAT CTA MT CAG
Gln Ala Leu Cys Leu Trp Leu Asn Ile Lys
Thr Lys Asp Leu Asn Gln
2 35 40 45
5
CTG CGG ATC ATG GGC ACC GTG CTG GGC ATT 549
ATC MG TTC CTA TCA GAC
Leu Arg Ile llet Gly Thr Val Leu Gly Ile
Ile Lys Phe Leu Ser Asp
50 55 60 65
GGC TGG CCA GTG AM GM ATC CCC TCC TAC 597
CCT CGG CCG TCC MG GGC
3 Gly Trp Pro Val Lys Glu Ile Pro Ser Tyr
0 Pro Arg Pro Ser Lys Gly
70 75 80
GAG CCA GAG GAC GAG GTC GAG GAC ACG CTG 645
GAG GTT GAG CTG AGG G11G
Glu Pro Glu Asp Glu Val Glu Asp Thr Leu
Glu Val Glu Leu Arg Glu
85 90 95
3 GAG AGC GGG ACG GAG GAG AGT GAC GAG AGG 693
5 GAG CCA ACC CCC AGT CCG
Glu Ser Gly Thr Glu Glu Ser Asp Glu Arg
Glu Pro Thr Pro Ser Pro
100 105 110
GTG CCA GAG CTC AGG CTG TCC ACA GAC TCC 741
ACC ATC TTG GAC AGT CGC
Val Pro Glu Leu Arg Leu Ser Thr Asp Ser
Thr lle Leu Asp Ser Arg
4 115 120 125
0
CAG GGC TGC GTC TCC AGG MG CTG GAG AGG CTC GAG TCA GAG GAA GAT 789
Gln Gly Cys Val Ser Arg Lys Leu Glu Arg Leu Glu Ser Glu Glu Asp
130 135 140 145
TCC ATA GGC TGG GGG ACA GCG GAC TGT GGC CCT GM GCC AGC AGG CAT 837
4 5 Ser Ile Gly Trp Gly Thr Ala Asp Cys Gly Pro Glu Ala Ser Arg His
150 155 160
TGT TTG ACT TCT ATC TAT AGA CCA TTC GTG GAC MA GCA CTG MG CM 885
Cys Leu Thr Ser Ile Tyr Arg Pro Phe Yal Asp Lys Ala Leu Lys Gln
165 170 175
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ATG GGG CTA AGA MG TTA ATT TTA CGA CTT 933
CAT MG CTT ATG MT GGG
Net Gly Leu Arg Lys Leu Ile Leu Arg Leu
His Lys Leu Net Asn Gly
180 185 190
TCC TTG CM AGA GCT CGT GTA GCT CTG GTG 981
MG GAC GAC CGT CCA GTG
Ser Leu Gln Arg Ala Arg Yal Ala Leu Val
Lys Asp Asp Arg Pro Val
195 200 205
GAG TTC TCT GAC TTT CCA GGT CCC ATG TGG 1029
GGC TCG GAT TAT GTG CAG
Glu Phe Ser Asp Phe Pro Gly Pro Net Trp
Gly Ser Asp Tyr Val Gln
210 215 220 225
1 TTG TCG GGA ACA CCT CCT TCC TCA GAG CAG 1077
O MG TGT AGC GCT GTG TCC
Leu Ser Gly Thr Pro Pro Ser Ser Glu Gln
Lys Cys Ser Ala Vel Ser
230 235 240
TGG GAA GAA CTG AGA GCC ATG GAC CTG CCT 1125
TCC TTT GAG CCC GCC TTC
Trp Glu Glu Leu Arg Ala Net Asp Leu Pro
Ser Phe Glu Pro Ala Phe
245 250 255
CTG GTG CTC TGT CGG GTC CTG CTG MC GTG 1173
ATC CAC GAG TGC CTG MG
Leu Val Leu Cys Arg Val Leu Leu Asn Val
Ile His Glu Cys Leu Lys
260 265 270
CTG CGG CTG GM CAG AGG CCT GCC GGG GAG 1221
CCT TCC CTC TTG AGT ATC
2 Leu Arg Leu Glu Gln Arg Pro Ala Gly Glu
0 Pro Ser Leu Leu Ser Ile
275 280 285
AM CAG CTA GTG CGA GAG TGT MA GAG GTC CTA 1269
MG GGC GGG CTC CTG
Lys Gln Leu Val Arg Glu Cys Lys Glu Val
Leu Lys Gly Gly Leu Leu
290 295 300 305
2 ATG MG CAG TAT TAC CAG TTC ATG CTG CAG 1317
5 GAG GTC CTG GGC GGA CTG
Net Lys Gln Tyr Tyr Gln Phe Net Leu Gln
Glu Val Leu Gly Gly Lau
310 315 320
GAG MG ACC GAC TGC MC ATG GAT GCC TTT GAG 1365
GAG GAC CTG CAG MG
Glu Lys Thr Asp Cys Asn Met Asp Ala Phe
Glu Glu Asp Leu Gln Lys
3 325 330 335
0
ATG CTG ATG GTG TAT TTT GAT TAC ATG AGA 1413
AGC TGG ATC CM ATG CTA
Met Leu Net Val Tyr Phe Asp Tyr Net Arg
Ser Trp Ile Gln Net Leu
340 345 350
CAG CAG TTA CCT CAG GCT TCC CAT AGC TTA 1461
AM MC CTG CTA GAA GAG
3 Gln Gln Leu Pro Gln Ala Ser His Ser Leu
5 Lys Asn Leu Leu Glu Glu
355 360 365
GM TGG MT TTC ACC AM GAA ATA ACC CAT TAT 1509
ATC CGT GGC GG11 GM
Glu Trp Asn Phe Thr Lys Gtu Ile Thr His
Tyr Ile Arg Gly Gly Glu
370 375 380 385
4 GCG CAG GCT GGA MG CTT TTC TGT GAC ATC 1557
O GCA GGG ATG CTG CTG AAA
Ala Gln Ala Gly Lys Leu Phe Cys Asp Ile
Ala Gly Net Leu Leu Lys
390 395 400
TCC ACA GGG AGC TTT CTG GM TCC GGC CTG 1605
CAG GAG AGC TGT GCT GAG
Ser Thr Gly Ser Phe Leu Glu Ser Gly Leu
Gln Glu Ser Cys Ala Glu
4 405 410 415
5
CTG TGG ACC AGC GCC GAC GAC MC GGT GCT 1653
GCC GAC GAG CTA AGG AGA
Leu Trp Thr Ser Ala Asp Asp Asn Gly Ala
Ala Asp Glu Leu Arg Arg
420 425 430
TCT GTC ATC GAG ATC AGC CGA GCA CTC MG 1701
GAG CTC TTC CAC GM GCC
5 Ser Val Ile Glu Ile Ser Arg Ala Leu Lys
0 Glu Leu Phe Nis Glu Ala
435 440 445
AGG GM AGA GCC TCC MG GCC CTG GGC TTT GCT 1749
AM ATG CTG AGG MG
Arg Glu Arg Ala Ser Lys Ala Leu Gly Phe
Ala Lys Net Leu Arg Lys
450 455 460 465
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GAC CTA GM ATA GCA GCA GAG TTC GTG CTA 1797
TCT GCA TCA GCC CGA GAG
Asp Leu Glu Ile Ala Ala Glu Phe Val Leu
Ser Ala Ser Ala Arg Glu
470 475 480
CTC CTG GAC GCT CTG AM GCA MG CAG TAT 1845
GTT MG GTA CAG ATT CCC
Leu Leu Asp Ala Leu Lys Ala Lys Gln Tyr
Val Lys Val Gln Ile Pro
485 490 495
GGG TTA GAG MT TTG CAC GTG TTT GTC CCC 1893
GAC AGC CTC GCT GAG GAG
Gly Leu Glu Asn Leu His Vel Phe Vel Pro
Asp Ser Leu Ala Glu Glu
500 505 510
1 MG AM ATT ATT TTG CAG CTA CTC MT GCT GCC 1941
O ACA GGA MG GAC TGC
Lys Lys lie Ile Leu Gln Leu Leu Asn Ala
Ala Thr Gly Lys Asp Cys
515 520 525
TCA MG GAT CCA GAC GAC GTC TTC ATG GAT 1989
GCC TTC CTG CTC CTG ACC
Ser Lys Asp Pro Asp Asp Yal Phe lief Asp
Ala Phe Leu Leu Leu Thr
1 530 535 540 545
5
MG CAT GGG GAC CGA GCC CGT GAC TCA GM 2037
GAT GGC TGG GGC ACA TGG
Lys His Gly Asp Arg Ala Arg Asp Ser Glu
Asp Gly Trp Gly Thr Trp
550 555 560
GAA GCT CGG GCT GTC AM ATT GTG CCT CAG 2085
GTG GAG ACT GTG GAC ACC
2 Glu Ala Arg Ala Vat Lys Ile Val Pro Gln
0 Yal Glu Thr Val Asp Thr
565 570 575
CTG AGA AGC ATG CAG GTG GAC MC CTT CTG 2133
CTG GTT GTC ATG GAG TCT
Leu Arg Ser het Gln Val Asp Asn Leu Leu
Leu Val Val Net Glu Ser
580 585 590
2 GCT CAC CTC GTA CTT CAG AGA AM GCC TTC 2181
5 CAG CAG TCC ATT GAG GGG
Ale His Leu Val Leu Gln Arg Lys Ala Phe
Gln Gln Ser Ile Glu Gly
595 600 605
CTG ATG ACT GTA CGC CAT GAG CAG ACA TCT 2229
AGC CAG CCC ATC ATC GCC
Leu Met Thr Val Arg His Glu Gln Thr Ser
Ser Gln Pro Ile 1le Ala
3 610 615 620 625
0
AM GGT TTG CAG CAG CTC MG MC GAT GCA CTT 2277
GAG CTA TGC MC AGA
Lys Gly Leu Gln Gln Leu Lys Asn Asp Ala
Leu Glu Leu Cys Asn Arg
630 635 640
ATC AGC GAT GCC ATC GAC CGT GTG GAC GC 2325
ATG TTC ACC CTG GAG TTC
3 Ile Ser Asp Ala 1le Asp Arg Yal Asp His
5 lief Phe Thr Leu Glu Phe
645 650 655
GAT GCT GAG GTC GAG GAG TCT GAG TCG GCC 2373
ACG CTG CAG CAG TAC TAC
Asp Ala Glu Val Glu Glu Ser Glu Ser Ale
Thr Leu Gln Gln Tyr Tyr
660 665 670
4 CGA GM GCC ATG ATT CAG GGC TAC MC TTT 2421
O GGG TTT GAG TAT CAT AM
Arg Glu Ale Met Ile Gln Gly Tyr Asn Phe
Gly Phe Glu Tyr His Lys
675 680 685
GAA GTT GTT CGT TTG ATG TCT GGG GAA TTC 2469
AGG CAG MG ATA GGA GAC
Glu Val Vat Arg Leu IAet Ser Gly Glu Phe
Arg Gln Lys Ile Gly Asp
4 690 695 700 705
5
MA TAT ATA AGC TTC GCC CAG MG TGG ATG 2517
MT TAC GTG CTG ACC AM
Lys Tyr Ile Ser Phe Ala Gln Lys Trp lief
Asn Tyr Yal Leu Thr Lys
710 715 720
TGC GAG AGC GGC AGA GGC ACA AGA CCC AGA 2565
TGG GCC ACC CM GGA TTT
5 Cys Glu Ser Gly Arg Gly Thr Arg Pro Arg
0 Trp Ala Thr Gln Gly Phe
725 730 735
GAT TTC CTA CM GCC ATT GAA CCT GCC TTT 2613
ATT TCA GCT TTA CCA GAA
Asp Phe Leu Gln Ala Ile Glu Pro Ala Phe
lie Ser Ala Leu Pro Glu
740 745 750
2~~6526
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GAT GAC TTC TTG AGT TTG CM 2661
GCC CTG ATG MT GAG TGC ATC
GGG GC
Asp Asp Phe Leu Ser Leu Gln
Ala Leu Met Asn Glu Cys Ile
Gly His
755 760 765
GTC ATA GGA MG CG GC AGC CCT 2709
GTC ACA GCT ATC GT CGG MC
AGC
Val Ile Gly Lys Pro His Ser
Pro Val Thr Ala Ile His Arg
Asn Ser
no 775 7ao 785
CCC CGC CCT GTG MG GTG CCC 2757
CGA TGC GC AGT G11C CCT CCT
MC CCT
Pro Arg Pro Val Lys Val Pro
Arg Cys His Ser Asp Pro Pro
Asn Pro
790 795 800
1 GC CTC ATC ATC CCG ACT CG 2805
O GAG GGA TTC AGC ACC CGG AGC
GTG CCT
His Leu Ile Ile Pro Thr Pro hr Ara Ser
Glu Gly Phc Ser T Val Pro
805 810 . 815
TCC GAC GCT CGG ACC CAT GGC 2853
MC TCT GTT GCT GCT GCT GCT
GCT GTT
Ser Aso Ala Arg Thr His Glv
Asn Ser Val Ala Ala Ala Ale
Ala Val
820 825 830
CGT GCC GCC GCC ACC ACT GCT 2901
GCT GGC CGC CCT GGC CCA GGT
GGT GGT
A~9 Ala Ala Ala Thr Thr Ala
Ala Glv Arg Pro Glv Pro Glv
Glv Glv
835 840 845
GAC TCT GTG CCA GCC MA CCT 2949
GTC MC ACT GCC CCT GAT ACC
AGG GGT
2 Asc Ser Val Pro Ala Lvs Pro
0 Val Asn Thr Ala Pro Aso Thr
Arg Gly
850 855 860 865
TCC AGT GTC CCT GAA MC GAC 2997
CGC TTG GCC TCC ATA GCT GG
GAA CTG
Ser Ser Val Pro Glu Asn Asp
Arg Leu Ala Ser Ile Ala Ala
Glu Leu
870 875 880
2 GG TTC AGG TCT CTG AGT CGG 3045
5 GC TG AGC CCC ACG GM GAG
CGA GAC
Gln Phe Arg Ser Leu Ser Arg
His Ser Ser Pro Thr Glu Glu
Arg Asp
885 890 895
GAG CG GCG TAT CCT CGG AGT 3093
GAC TG AGT GGA TG ACT CGG
AGA AGC
Glu Pro Ala Tyr Pro Arg Ser
Asp Ser Ser Gly Ser Thr Arg
Arg Ser
3 900 905 910
0
TGG GM CTT CGI1 AG CTC ATC 3141
AGC GG ACC AM GAC TCG GCC
TCT MG
Trp Glu Leu Arg Thr Leu Ile
Ser Gln Thr Lys Asp Ser Ala
Ser Lys
915 920 925
GG GGG CCC ATA GM GCT ATC 3189
GG MG TG GTC CGA CTG TTT
GM GAG
3 Gln Gly Pro Ile Glu Ala Ile
5 Gln Lys Ser Val Arg Leu Phe
Glu Glu
930 935 940 945
AGG AGG TAT CGA GAG ATG AGG 3237
AGA MG MT ATC ATC GGC GA
GTG TGC
Arg Arg Tyr Arg Glu lief Arg
Arg Lys Asn lie Ile Gly Gln
Val Cys
950 955 960
4 GAT ACC CCT MG TCC TAT GAT 3285
O MC GTC ATG GT GTT GGA CTG
AGG MG
Asp Thr Pro Lys Ser Tyr Asp
Asn Val Ref His Val Gly Leu
Arg Lys
965 970 975
GTG AG TTT MG TGG CM AGA GGA 3333
MC MA ATT GGA GM GGA GG TAT
Val Thr Phe Lye Trp Gln Arg
Gly Asn Lys Ile Gly Glu Gly
Gln Tyr
4 980 985 990
5
GGA AM GTA TAC ACC TGC ATC 3381
AGT GTT GAC AG GGG GAG CTG
ATG GCC
Gly Lys Val Tyr Thr Cys Ile
Ser Val Asp Thr Gly Glu Leu
Met Ala
995 1000 1005
ATG MG GAG ATT CGA TTT GG 3429
CCT MC GAC GC MG ACT ATC
MG GAG
5 Ret Lys Glu Ile Arg Phe Gln
0 Pro Asn Asp His lys Thr ile
Lys Glu
1010 1015 1020 1025
ACT GCA GAC GAG TTG AM ATA 3477
TTT GM GGC ATC MG GC CCC
MC CTG
Thr Ala Asp Glu Leu Lys Ile
Phe Glu Gly Ile Lys His Pro
Asn Leu
1030 1035 1040
2i~b5~6
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GTC CGG TAT TTT GGC GTG GAG CTT GC AGG 3525
GM GAG ATG TAC ATC TTC
Yal Arg Tyr Phe Gly Val Glu Leu His Arg
Glu Glu Met Tyr Ile Phe
1045 1050 1055
ATG GAG TAC TGT GAT GAG GGT ACA CTA GAG 3573
GAG GTG TG CGA CTG GGC
Ilet Glu Tyr Cys Asp Glu Gly Thr Leu Glu
Glu Val Ser Arg Leu Gly
1060 1065 1070
CTG CAG GAG GC GTC ATC AGG TTA TAT ACC 3621
MG GG ATC ACT GTC GCC
Leu Gln Glu His Val Ile Arg Leu Tyr Thr
Lys Gln Ile Thr Val Ala
1075 1080 1085
Z ATC MC GTC CTC CAT GAG CAC GGC ATC GTT 3669
O GC CGA GAC ATC AM GGT
Ile Asn Val Leu His Glu His Gly 1le Val
His Arg Asp Ile Lys Gly
1090 1095 1100 1105
GCC MT ATC TTC CTT ACG TG TCT GGA CTA ATC 3717
MG CTG GGA GAT TTT
Ala Asn Ile Phe Leu Thr Ser Ser Gly Leu
Ile Lys Leu Gly Asp Phe
1 1110 1115 1120
5
GGA TGC TCT GTA AM CTT AM MC MC GCC CAG 3765
ACC ATG CCC GGJ1 GAG
Gly Cys Ser Val Lys Leu Lys Asn Asn Ala
Gln Thr ltet Pro Gly Glu
1125 1130 1135
GTG MC AGC ACC CTA GGG AG GG GCT TAC ATG 3813
GCC CCT GM GTT ATT
2 Val Asn Ser Thr Leu Gly Thr Ala Ala Tyr
0 llet Ala Pro Glu Val 1le
,
1140 1145 1150
ACC CGA GCC AM GGA GM GGC GC GGA CGT GCG 3861
GG GAT ATC TGG AGT
Thr Arg Ala Lys Gly Glu Gly His Gly Arg
Ala Ala Asp ile Trp Ser
1155 1160 1165
2 CTG GGG TGC GTC GTC ATA GAG ATG GTG ACT 3909
5 GGC MG CGG CCT TGG GT
Leu Gly Cys Val Val Ile Glu Net Val Thr
Gly Lys Arg Pro Trp His
1170 1175 1180 1185
GAG TAT GM GC MC TTT GG ATT ATG TAC MG 3957
GTG GGG ATG GGA GC
Glu Tyr Glu His Asn Phe Gln Ile llet Tyr
Lys Val Gly liet Gly His
3 1190 1195 1200
0
MG CG CG ATC CCG GM AGG CTA AGC CCT GM 4005
GGA MG GCC TTT CTC
Lys Pro Pro Ile Pro Glu Arg Leu Ser Pro
Glu Gly Lys Ala Phe leu
1205 1210 1215
TCG GC TGC CTG GM AGT GAC CCG MG ATA CGG 4053
TGG AG GCC AGC GG
3 Ser His Cys Leu Glu Ser Asp Pro Lys lle
5 Arg Trp Thr Ala Ser Gln
1220 1225 1230
CTC CTC GAC GC GCT TTT GTC MG GTT TGC AG 4095
GAT GM GAG
Leu Leu Asp His Ala Phe Val Lys Val Cys
Thr Asp Glu Glu
1235 1240 1245
4 TGMGTGMC GGTCCGTGG CCTAGTAGTG TGTGGAGGA 4155
O ATCCCGTGAT GCTACTGTA
TGTMTATTT ACATAMGAC TGGGCGCAG GCGGCCTTCC 4215
TMCCTCCG GGACTGAAGA
CTAGGGGGT GAGAGCCTC ACTTCTGCTG CTCCTGTCGC 4275
CTGCTGAGTG AGGTGCTGA
GGTTAMGGA GCCGGCGTT MGTGCCATT ACTACTGTAC 4335
ACGGCGCCG CCTCTGTCCC
CTCCGACCCT CTCGTGACTG AGAACGACC GTGTGTGG 4395
GGGTGTTT TTGAGCTCCT
4 GGGGTTGGA AGMGTGTA GTGTTCCCGG GTGTCCGGGA 4455
5 CGTTTATTTC MCCTCCTGG
TCGTTGGCTC TGACTGTGGA GCCTCCTTGT TCGAMGCTG4515
GGGTTTGTT ATGGMGGC
TCGTMGTGA AGCTGMGM MGGTTCTTT TTCMTAMT GGTTTATTTT4575
AGGAMGCGA
AAAMAAAM AAAMM 4592
MEKK 5 represents a splice variant of MEKK
4. The splice
50 insert is shown by the underlined portion of the sequence
shown in Table 5.
CA 02186526 1999-08-20
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The amino acid sequences for MEKK 2 and MEKK 3 compared with
the amino acid sequence of MEKK 1 are shown in Table 6.
Table 6.
MVTAVPAVFSKLVTMLMASGSTHFTRMRRRLMAIADEVEIAEVIQLGVEDTVDGHQDSL MEKK 1
MDDQQALNSIMQDI--------- 2
MDEQEALDSIMKDLVALQMSRRTRL- 3
AVAPTSCLENSSLEHTVHREKTGKGLSATRLSASSEDISDRLAGVSVGLPSSTTTEQPKP 1
AVLHKPVGQHYLYKKPGRQNLHHQKNRMMFESNLNIEEEKRILQVTRPVKLEDLRSKSKI 2
S--GYETMKNRDTGHPNRQSDVRIKFENNGERRI-IAFSRPVRYEDVEHKVTTVFGQPLD 3
AVQTKGRPHSQCLNSS-PLSHAQLMFPAPSAPCSSAPSVPDISKHRPQAFVPCKIPSASP 1
AFGQSMDLHYTNNELVIPLTTQDDLDKAVELLDRSIHMKSL-KILLVVNGSTQA-TNLEP 2
LHYMNNELSILLKNQDDLDKAIDILDRSSSMKSLRILLLSQDRNHTSSSPHSGVSRQVRI 3
QTQRKFSLQFQRNCSEHRDSDQLSPVFTQS-RPPPSSNIHRPKPSRPVPGSTSKLGDATK 1
SPSPEDLNNTPLGAERKKRLSVVGPPNR--DRSSPPPGYIPDILHQIARNGSFTSINSEG 2
KPSQSAGDINTIYQAPEPRSRHLSVSSQNPGRSSPPPGYVPERQQHIARQGSYTSINSEG 3
SSMTLDLGSASRCDDSFGGGGNSGNAVIPSDETVFTPVEDKCRLDVNTELNSSIEDLLEA 1
EFIPESMDQ-MLDPLSLSSPENSGSGSCPSLDSPLDGESYPKSRMPRAQSYPDNHQEFTD 2
EFIPETSEQCMLDPLSSAENSLSGSCQSLDRSADSPSFRKSQMSRARSFPDNR---ECSD 3
SMPSSDTTVTFKSEVAVLSPEKAENDDTYKDDVNHNQKCKEKMEAEEEEALAIAMAMSAS 1
YDNPIFEKFGKGGTYPRRYHVSYHHQEYNDGRKTFPRARRTQGTSFRSPVSFSPTDHSLS 2
K----RETQLYDKGVKGGTYPRRYHVSVHHKDYNDGRRTFPRIRRHQGNLFTLVPSSRSL 3
QDALPIVPQLQVENGEDIIIIQQDTPETLPGHTKAKQPYREDAEHTLKGQQIGLGAFSSCY 1
TSSGSSVFTPEYDDSRIRRRGSDIDNPTLTVTDISPPSRSPRAPTNV~1RLGKLLGQGAFGR 2
STNGENMGVAVQYLDPRGRLRSADSENALTVQERNVPTKSPSAPINWRRGKLLGQGAFGR 3
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QAQDVGTGTLMAVKQVTYVRNTSSE EEVVEALREEIRMMGHLNHPNIIRMLGATCEKSN 1
VYLCYDVDTGRELAVKOVQFNPESPETSKEVNALECEIQLLKNLLHERIVQYYGCLRDPQ 2
VYLCYDVDTGRELASKOVQFDPDSPETSREVSALECEIQLLKNLQHERIVQYYGCLRDRA 3
YNLFIEWMAGGSVAHLLSKYGAFKESVVINYTEQLLRGLSYLHEN--Q-IIHRDVKGANL 1
EKTLSIFMELSPGGSIKDQLKAYGALTENVTRKYTRQILEGVHYLHSNMIVHRDIKGANI 2
EKILTIFMEYMPGGSVKDQLKAYGALTESVTRKYTRQILEGMSYLHSNMIVHRDIKGANI 3
LIDSTGQ-RLRIADFGAAARLASR-GTGAGEFQGQLLGTIAFMAPEVLRGQQYGRSCD'VVJ 1
LRDSTGNIRLGDFGASKRLQTICLSGTGMKSVTG-PY----WMSPEVISGEGYGRKADIV~I 2
LRDSAGNVKLGDFGASKRL TICMSGTGIRSVTGTPY----WMSPEVISGEGYGRKADVHI 3
SVGCAIIEMACAKPPWNAEKHSNHLALIFKIASATTAPSIPSHLSPGLRDVAVRCLELQP 1
SVACTWEMLTEKPPHT-AEFEA-MAA-IFKIATQPTNPKLPPHVSDYTRDFLKRIFVEAK 2
SLGCTWEMLTEKPPVJ-AEYEA-MAA-IFKIATQPTNPQLPSHISEHGRDFLRRIFVEAR 3
ODRPPSRE-LLKHPVFRTTV~I
L-RP-SAEELLRHMFVHYH
Q-RP-SAEELLTHHFA LVY
Bold Amino Terminus- Regulatory Domain
Underline seauence- Regulatory hinge Sequence
Bold Italics- Catalytic Domain
Table 7 shows the amino acid sequence of the kinase domain
of MEKK 4 compared with the kinase domains of MEKK 1, MEKK 2 and
MEKK 3.
Table 7.
.IRFQPNDHKTIKETADEELKIFEGIKHPNLVRYFGV..ELHREEM.YI MEKK4
TYVRNTSSEQEEWEALREEIRMMGHLNHPNIIRMLGATCEKSNYNLFIE MEKK1
QVQFNPESPETSKEVNALECEIQLLKNLLHERIVQYYGCLRDPQEKTLSI MEKK2
QVQFDPDSPETSKEVSALECEIQLLKNLQHERIVQYYGCLRDRAEKILTI MEKK3
CA 02186526 1999-08-20
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IFMEYCDEGTLEEVSRLGLQEHV.I.RLYTKQITVAINVLHEHGNV MEKK4
WMAGGSVAHLLSKYGAFKESW .IN..YTEQLLRGLSYLHENQII MEKK1
FMELSPGGSIKDQLKAYGALTENVTRKYTRQILEGVHYLHSNMIV MEKK2
FMEYMPGGSVKDQLKAYGALTESVTRKYTRQILEGMSYLHSNMIV MEKK3
The foregoing SEQ ID NO's represent sequences deduced
according to methods disclosed in the Examples. It should be
noted that since nucleic acid and amino acid sequencing
technology is not entirely error-free, the foregoing SEQ ID NO' s,
at best represent apparent nucleic acid and amino acid sequences
of MEKK proteins of the present invention.
According to the present invention, an MEKK protein of the
present invention can include MEKK proteins that have undergone
post-tranlational modification. Such modification can include,
for example, glycosylation (e. g., including addition of N-linked
and/or O-linked oligosaccharides) or post-translational
conformational changes or post-translational deletions.
Another embodiment of the present invention is an isolated
nucleic acid molecule capable of hybridizing, under stringent
conditions, with an MEKK protein gene encoding an MEKK protein
of the present invention. In accordance with the present
invention, an isolated nucleic acid molecule is a nucleic acid
molecule that has been removed from its natural milieu (i.e. ,
that has been subject to human manipulation) . As such, "isolated"
does not reflect the extent to which the nucleic acid molecule
WO 95/28421 PCTILTS94/11690
2i~b526
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has been purified. An isolated nucleic acid molecule can
include DNA, RNA, or derivatives of either DNA or RNA.
An isolated nucleic acid molecule of the present
invention can be obtained from its natural source either as
an entire (i.e., complete) gene or a portion thereof
capable of forming a stable hybrid with that gene. As used
herein, the phrase "at least a portion of" an entity refers
to an amount of the entity that is at least sufficient to
have the functional aspects of that entity. For example,
at least a portion of a nucleic acid sequence, as used
herein, is an amount of a nucleic acid sequence capable of
forming a stable hybrid with a particular desired gene
(e. g., MEKK genes) under stringent hybridization
conditions. An isolated nucleic acid molecule of the
present invention can also be produced using recombinant
DNA technology (e. g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Isolated
MEKK protein nucleic acid molecules include natural nucleic
acid molecules and homologues thereof, including, but not
limited to, natural allelic variants and modified nucleic
acid molecules in which nucleotides have been inserted,
deleted, substituted, and/or inverted in such a manner that
such modifications do not substantially interfere with the
nucleic acid molecule's ability to encode an MEKK protein
of the present invention or to form stable hybrids under
stringent conditions with natural nucleic acid molecule
isolates of MEKK.
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Preferred modifications to an MEKK protein nucleic
acid molecule of the present invention include truncating
a full-length MEKK protein nucleic acid molecule by, for
example: deleting at least a portion of an MEKK protein
nucleic acid molecule encoding a regulatory domain
(examples illustrated in Table 6) to produce a
constitutively active MEKK protein; deleting at least a
portion of an MEKK protein nucleic acid molecule encoding
a catalytic domain (examples illustrated in Table 6) to
produce an inactive MEKK protein; and modifying the MEKK
protein to achieve desired inactivation and/or stimulation
of the protein, for example, substituting a codon encoding
a lysine residue in the catalytic domain (i.e.,
phosphotransferase domain) with a methionine residue to
inactivate the catalytic domain.
A preferred truncated MERX nucleic acid molecule
encodes a form of an MEKK protein containing a catalytic
domain but that lacks a regulatory domain. Preferred
catalytic domain truncated MEKK nucleic acid molecules
encode residues from about 352 to about 672 of MEKK 1, from
about 352 to about 619 of MEKK 2, from about 358 to about
626 of MEKK 3, from about 811 to about 1195 of MEIQC 4 or
from about 863 to about 1247 of MEKK 5.
Another preferred truncated MEKK nucleic acid molecule
encodes a form of an MEKK protein comprising an NHZ-terminal
regulatory domain a catalytic domain but lacking a
catalytic domain. Preferred regulatory domain truncated
MEKK nucleic acid molecules encode residues from about 1 to
~18o5t~'~
_._ WO 95/28421 PCT/US94/11690
-47-
about 3 69 f or MEKK 1, from about 1 to about 3 3 5 f or MEKK 2 ,
from about 1 to about 3 6 0 f or MEKK 3 , from about 1 to about
8 2 5 f or MEKK 4 and from about 1 to about 8 7 5 f or MEKK 5 ,
thereby removing the regulatory domain to form the
truncated MEKK molecule.
An isolated nucleic acid molecule of the present
invention can include a nucleic acid sequence that encodes
at least one MEKK protein of the present invention,
examples of such proteins being disclosed herein. Although
the phrase "nucleic acid molecule" primarily refers to the
physical nucleic acid molecule and the phrase "nucleic acid
sequence" primarily refers to the sequence of nucleotides
that comprise the nucleic acid molecule, the two phrases
can be used interchangeably. As heretofore disclosed, MEKK
proteins of the present invention include, but are not
limited to, proteins having full-length MEKK protein coding
regions, portions thereof, and other MEKK protein
homologues.
As used herein, an MEKK protein gene includes all
nucleic acid sequences related to a natural MEKK protein
gene such as regulatory regions that control production of
an MEKK protein encoded by that gene (including, but not
limited to, transcription, translation or post-translation
control regions) as well as the coding region itself. A
nucleic acid molecule of the present invention can be an
isolated natural MEKK protein nucleic acid molecule or a
homologue thereof. A nucleic acid molecule of the present
invention can include one or more regulatory regions, full-
~~ ~~5z~
WO 95/28421 PCT/US94/11690
-48-
length or partial coding regions, or combinations thereof.
The minimal size of an MEKK protein nucleic acid molecule
of the present invention is the minimal size capable of
forming a stable hybrid under stringent hybridization
conditions with a corresponding natural gene.
An MEKK protein nucleic acid molecule homologue can be
produced using a number of methods known to those skilled
in the art (see, e.g., Sambrook et al., ibid.). For
example, nucleic acid molecules can be modified using a
variety of techniques including, but not limited to,
classic mutagenesis techniques and recombinant DNA
techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment,
ligation of nucleic acid fragments, polymerise chain
reaction (PCR) amplification and/or mutagenesis of selected
regions of a nucleic acid sequence, synthesis of
oligonucleotide mixtures and ligation of mixture groups to
"build" a mixture of nucleic acid molecules and
combinations thereof. Nucleic acid molecule homologues can
be selected from a mixture of modified nucleic acids by
screening for the function of the protein encoded by the
nucleic acid (e.g., the ability of a homologue to
phosphorylate MEK protein or JEK protein) and/or by
hybridization with isolated MEKK protein nucleic acids
under stringent conditions.
One embodiment of the present invention is an MEKK
protein nucleic acid molecule capable of encoding at least
__? 1 ~365a6
WO 95/28421 PCT/US94/11690
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a portion of an MEKK protein, or a homologue thereof, as
described herein. A preferred nucleic acid molecule of the
present invention includes, but is not limited to, a
nucleic acid molecule that encodes a protein having at
least a portion of an amino acid sequence represented by
SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8 and SEQ
ID NO:10, or homologues thereof.
A preferred nucleic acid molecule of the present
invention is capable of hybridizing under stringent
conditions to a nucleic acid that encodes at least a
portion of an MEKK protein, or a homologue thereof. Also
preferred is an MEKK protein nucleic acid molecule that
includes a nucleic acid sequence having at least about 50%,
preferably at least about 75%, and more preferably at least
about 85% homology with the corresponding regions) of the
nucleic acid sequence encading the catalytic domain of an
MEKK protein, or a homologue thereof . Also preferred is an
MEKK protein nucleic acid molecule that includes a nucleic
acid sequence having at least about 20%, preferably at
least about 30%, and more preferably at least about 40%
homology with the corresponding regions) of the nucleic
acid sequence encoding the NH2-terminal regulatory domain of
an MEKK protein, or a homologue thereof. A particularly
preferred nucleic acid sequence is a nucleic acid sequence
having at least about 50%, preferably at least about 75%,
and more preferably at least about 85% homology with a
nucleic acid sequence encoding the catalytic domain of an
amino acid sequence represented by SEQ ID N0:2, SEQ ID
i
21~~526
WO 95/28421 PCT/US94/11690
-50-
N0:4, SEQ ID N0:6, SEQ ID N0:8 and SEQ ID NO:10. Another
particularly preferred nucleic acid sequence is a nucleic
acid sequence having at least about 20%, preferably at
least about 30%, and more preferably at least about 40%
homology with a nucleic acid sequence encoding the NHZ-
terminal regulatory domain of an amino acid sequence
represented by SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ
ID NO:8 and SEQ ID NO:10.
Such nucleic acid molecules can be a full-length gene
and/or a nucleic acid molecule encoding a full-length
protein, a hybrid protein, a fusion protein, a multivalent
protein or a truncation fragment. More preferred nucleic
acid molecules of the present invention comprise isolated
nucleic acid molecules having a nucleic acid sequence as
represented by SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5, SEQ
ID N0:7 or SEQ ID N0:9.
Knowing a nucleic acid molecule of an MEKK protein of
the present invention allows one skilled in the art to make
copies of that nucleic acid molecule as well as to obtain
additional portions of MEKK protein-encoding genes (e. g.,
nucleic acid molecules that include the translation start
site and/or transcription and/or translation control
regions), and/or MEKK protein nucleic acid molecule
homologues. Knowing a portion of an amino acid sequence of
an MEKK protein of the present invention allows one skilled
in the art to clone nucleic acid sequences encoding such an
MEKK protein.
WO 95128421 PCT/~JS94/11690
-51-
The present invention also includes nucleic acid
molecules that are oligonucleotides capable of hybridizing,
under stringent conditions, with complementary regions of
other, preferably longer, nucleic acid molecules of the
present invention that encode at least a portion of an MEKK
protein, or a homologue thereof. A preferred
oligonucleotide is capable of hybridizing, under stringent
conditions, with a nucleic acid molecule that is capable of
encoding at least a portion of an amino acid sequence
represented by SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ
ID NO: 8 and SEQ ID N0:10, or homologues thereof . A more
preferred oligonucleotide is capable of hybridizing to a
nucleic acid molecule having a nucleic acid sequence as
represented by SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5, SEQ
ID N0:7 and SEQ ID N0:9, or complements thereof.
Oligonucleotides of the present invention can be RNA,
DNA, or derivatives of either. The minimal size of such
oligonucleotides is the size required to form a stable
hybrid between a given oligonucleotide and the
complementary sequence on another nucleic acid molecule of
the present invention. Minimal size characteristics are
disclosed herein. The size of the oligonucleotide must
also be sufficient for the use of the oligonucleotide in
accordance with the present invention. Oligonucleotides of
the present invention can be used in a variety of
applications including, but not limited to, as probes to
identify additional nucleic acid molecules, as primers to
amplify or extend nucleic acid molecules or in therapeutic
1 F~65~6
WO 95/28421 PCT/ITS94/11690
-52-
applications to inhibit, for example, expression of MEKK
proteins by cells. Such therapeutic applications include
the use of such oligonucleotides in, for example,
antisense-, triplex formation-, ribozyme- and/or RNA drug-
s based technologies. The present invention, therefore,
includes use of such oligonucleotides and methods to
interfere with the production of MEKK proteins.
In one embodiment, an isolated MEKK protein of the
present invention is produced by culturing a cell capable
of expressing the protein under conditions effective to
produce the protein, and recovering the protein. A
preferred cell to culture is a recombinant cell that is
capable of expressing the MEKK protein, the recombinant
cell being produced by transforming a host cell with one or
more nucleic acid molecules of the present invention.
Transformation of a nucleic acid molecule into a cell can
be accomplished by any method by which a nucleic acid
molecule can be inserted into the cell. Transformation
techniques include, but are not limited to, transfection,
electroporation, microinjection, lipofection, adsorption,
and protoplast fusion. A recombinant cell may remain
unicellular or may grow into a tissue, organ or a
multicellular organism. Transformed nucleic acid molecules
of the present invention can remain extrachromosomal or can
integrate into one or more sites within a chromosome of the
transformed (i.e., recombinant) cell in such a manner that
their ability to be expressed is retained.
WO 95/28421 ~ ~ ~ PCT/US94/11690
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The present invention also includes a recombinant
vector which includes at least one MEKK protein nucleic
acid molecule of the present invention inserted into any
vector capable of delivering the nucleic acid molecule into
a host cell. Such a vector contains heterologous nucleic
acid sequences, for example nucleic acid sequences that are
not naturally found adjacent to MEKK protein nucleic acid
molecules of the present invention. The vector can be
either RNA or DNA, and either prokaryotic or eukaryotic,
and is typically a virus or a plasmid. Recombinant vectors
can be used in the cloning, sequencing, and/or otherwise
manipulating of MEKK protein nucleic acid molecules of the
present invention. One type of recombinant vector, herein
referred to as a recombinant molecule and described in more
detail below, can be used in the expression of nucleic acid
molecules of the present invention. Preferred recombinant
vectors are capable of replicating in the transformed cell.
Preferred nucleic acid molecules to insert into a
recombinant vector includes a nucleic acid molecule that
encodes at least a portion of an MEKK protein, or a
homologue thereof. A more preferred nucleic acid molecule
to insert into a recombinant vector includes a nucleic acid
molecule encoding at least a portion of an amino acid
sequence represented by SEQ ID NO: 2 , SEQ ID NO: 4 , SEQ ID
N0:6, SEQ ID N0:8 and/or SEQ ID NO:10, or homologues
thereof. An even more preferred nucleic acid molecule to
insert into a recombinant vector includes a nucleic acid
21 ~~52~
WO 95128421 PCT/US94/11690
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molecule represented by SEQ ID NO:1, SEQ ID N0:3, SEQ ID
N0:5, SEQ ID N0:7 and/or SEQ ID N0:9, or complements
thereof .
Suitable host cells for transforming a cell can
include any cell capable of producing MEKK proteins of the
present invention after being transformed with at least one
nucleic acid molecule of the present invention. Host cells
can be either untransformed cells or cells that are already
transformed with at least one nucleic acid molecule.
Suitable host cells of the present invention can include
bacterial, fungal (including yeast), insect, animal and
plant cells. Preferred host cells include bacterial, yeast,
insect and mammalian cells, with mammalian cells being
particularly preferred.
A recombinant cell is preferably produced by
transforming a host cell with one or more recombinant
molecules, each comprising one or more nucleic acid
molecules of the present invention operatively linked to an
expression vector containing one or more transcription
control sequences. The phrase operatively linked refers to
insertion of a nucleic acid molecule into an expression
vector in a manner such that the molecule is able to be
expressed when transformed into a host cell. As used
herein, an expression vector is a DNA or RNA vector that is
capable of transforming a host cell and of effecting
expression of a specified nucleic acid molecule.
Preferably, the expression vector is also capable of
replicating within the host cell. Expression vectors can
WO 95/28421 ~ 6 PCT/ITS94/11690
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be either prokaryotic or eukaryotic, and are typically
viruses or plasmids. Expression vectors of the present
invention include any vectors that function (i.e., direct
gene expression) in recombinant cells of the present
invention, including in bacterial, fungal, insect, animal,
and/or plant cells. As such, nucleic acid molecules of the
present invention can be operatively linked to expression
vectors containing regulatory sequences such as promoters,
operators, repressors, enhancers, termination sequences,
origins of replication, and other regulatory sequences that
are compatible with the recombinant cell and that control
the expression of nucleic acid molecules of the present
invention. As used herein, a transcription control
sequence includes a sequence which is capable of
controlling the initiation, elongation, and termination of
transcription. Particularly important transcription
control sequences are those which control transcription
initiation, such as promoter, enhancer, operator and
repressor sequences. Suitable transcription control
2o sequences include any transcription control sequence that
can function in at least one of the recombinant cells of
the present invention. A variety of such transcription
control sequences are known to those skilled in the art.
Preferred transcription control sequences include those
which function in bacterial, yeast, and mammalian cells,
such as, but not limited to, tac, lac, trp, trc, oxy-pro,
omp/ lpp, rrnB, bacteriophage lambda ( ~1 ) ( such as ~lp~ and ~lpR
and fusi~ons that include such promoters) , bacteriophage T7,
zi~~5z6
WO 95/28421 PCT/US94/11690
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T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage
SPO1, metallothionein, alpha mating factor, baculovirus,
vaccinia virus, herpesvirus, poxvirus, adenovirus, simian
virus 40, retrovirus actin, retroviral long terminal
repeat, Rous sarcoma virus, heat shock; phosphate and
nitrate transcription control sequences, as well as other
sequences capable of controlling gene expression in
prokaryotic or eukaryotic cells. Additional suitable
transcription control sequences include tissue-specific
promoters and enhancers as well as lymphokine-inducible
promoters (e.g., promoters inducible by interferons or
interleukins). Transcription control sequences of the
present invention can also include naturally occurring
transcription control sequences naturally associated with
a DNA sequence encoding an MEKK protein.
Preferred nucleic acid molecules for insertion into an
expression vector include nucleic acid molecules that
encode at least a portion of an MEKK protein, or a
homologue thereof. A more preferred nucleic acid molecule
for insertion into an expression vector includes a nucleic
acid molecule encoding at least a portion of an amino acid
sequence represented by SEQ ID NO: 2 , SEQ ID NO: 4 , SEQ ID
N0:6, SEQ ID N0:8 and/or SEQ ID NO:10, or homologues
thereof. An even more preferred nucleic acid molecule for
insertion into an expression vector includes a nucleic acid
molecule represented by SEQ ID NO:1, SEQ ID N0:3, SEQ ID
N0:5, SEQ ID N0:7 and/or SEQ ID N0:9, or complements
thereof .
W O 95/28421
PCT/US94/11690
-57-
Expression vectors of the present invention may also
contain fusion sequences which lead to the expression of
inserted nucleic acid molecules of the present invention as
fusion proteins. Inclusion of a fusion sequence as part of
an MEKK nucleic acid molecule of the present invention can
enhance the stability during production, storage and/or use
of the protein encoded by the nucleic acid molecule.
Furthermore, a fusion segment can function as a tool to
simplify purification of an MEKK protein, such as to enable
purification of the resultant fusion protein using affinity
chromatography. A suitable fusion segment can be a domain
of any size that has the desired function (e. g., increased
stability and/or purification tool). It is within the
scope of the present invention to use one or more fusion
segments. Fusion segments can be joined to amino and/or
carboxyl termini of an MEKK protein. Linkages between
fusion segments and MEKK proteins can be constructed to be
susceptible to cleavage to enable straight-forward recovery
of the MEKK proteins. Fusion proteins are preferably
produced by culturing a recombinant cell transformed with
a fusion nucleic acid sequence that encodes a protein
including the fusion segment attached to either the
carboxyl and/or amino terminal end of an MEKK protein.
A recombinant cell of the present invention includes
any cells transformed with at least one of any nucleic acid
molecule of the present invention. A preferred recombinant
cell is a cell transformed with at least one nucleic acid
molecule that encodes at least a portion of an MEKK
WO 95/28421 PCT/US94/11690
-58-
protein, or a homologue thereof. A more preferred
recombinant cell is transformed with at least one nucleic
acid molecule that is capable of encoding at least a
portion of an amino acid sequence represented by SEQ ID
NO: 2 , SEQ ID NO: 4 , SEQ ID NO: 6 SEQ ID NO: 8 and/or SEQ ID
NO:10, or homologues thereof. An even more preferred
recombinant cell is transformed with at least one nucleic
acid molecule represented by SEQ ID NO:1, SEQ ID N0:3, SEQ
ID N0:5, SEQ ID N0:7 and/or SEQ ID N0:9, or complements
thereof. Particularly preferred recombinant cells include
mammalian cells involved in a disease transformed with at
least one of the aforementioned nucleic acid molecules.
It may be appreciated by one skilled in the art that
use of recombinant DNA technologies can improve expression
of transformed nucleic acid molecules by manipulating, for
example, the number of copies of the nucleic acid molecules
within a host cell, the efficiency with which those nucleic
acid molecules are transcribed, the efficiency with which
the resultant transcripts are translated, and the
efficiency of post-translational modifications.
Recombinant techniques useful for increasing the expression
of nucleic acid molecules of the present invention include,
but are not limited to, operatively linking nucleic acid
molecules to high-copy number plasmids, integration of the
nucleic acid molecules into one or more host cell
chromosomes, addition of vector stability sequences to
plasmids, substitutions or modifications of transcription
control signals (e. g., promoters, operators, enhancers),
WO 95/28421 PCT/US94/11690
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substitutions or modifications of translational control
signals (e. g., ribosome binding sites, Shine-Dalgarno
sequences), modification of nucleic acid molecules of the
present invention to correspond to the codon usage of the
host cell, deletion of sequences that destabilize
transcripts, and use of control signals that temporally
separate recombinant cell growth from recombinant protein
production during fermentation. The activity of an
expressed recombinant protein of the present invention may
be improved by fragmenting, modifying, or derivatizing the
resultant protein.
As used herein, amplifying the copy number of a
nucleic acid sequence in a cell can be accomplished either
by increasing the copy number of the nucleic acid sequence
in the cell's genome or by introducing additional copies of
the nucleic acid sequence into the cell by transformation.
Copy number amplification is conducted in a manner such
that greater amounts of enzyme are produced, leading to
enhanced conversion of substrate to product. For example,
recombinant molecules containing nucleic acids of the
present invention can be transformed into cells to enhance
enzyme synthesis. Transformation can be accomplished using
any process by which nucleic acid sequences are inserted
into a cell. Prior to transformation, the nucleic acid
sequence on the recombinant molecule can be manipulated to
encode an enzyme having a higher specific activity.
In accordance with the present invention, recombinant
cells can be used to produce an MEKK protein of the present
WO 95/28421 U , PCT/US94/11690
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invention by culturing such cells under conditions
effective to produce such a protein, and recovering the
protein. Effective conditions to produce a protein
include, but are not limited to, appropriate media,
bioreactor, temperature, pH and oxygen conditions that
permit protein production. An appropriate, or effective,
medium refers to any medium in which a cell of the present
invention, when cultured, is capable of producing an MEKK
protein. Such a medium is typically an aqueous medium
comprising assimilable carbohydrate, nitrogen and phosphate
sources, as well as appropriate salts, minerals, metals and
other nutrients, such as vitamins. The medium may comprise
complex nutrients or may be a defined minimal medium.
Cells of the present invention can be cultured in
conventional fermentation bioreactors, which include, but
are not limited to, batch, fed-batch, cell recycle, and
continuous fermentors. Culturing can also be conducted in
shake flasks, test tubes, microtiter dishes, and petri
plates. Culturing is carried out at a temperature, pH and
oxygen content appropriate for the recombinant cell. Such
culturing conditions are well within the expertise of one
of ordinary skill in the art.
Depending on the vector and host system used for
production, resultant MEKK proteins may either remain
within the recombinant cell or be secreted into the
fermentation medium. The phrase "recovering the protein"
refers simply to collecting the whole fermentation medium
containing the protein and need not imply additional steps
WO 95128421 ~ J ,~ PCT/LTS94/11690
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of separation or purification. MEKK proteins of the
present invention can be purified using a variety of
standard protein purification techniques, such as, but not
limited to, affinity chromatography, ion exchange
chromatography, filtration, electrophoresis, hydrophobic
interaction chromatography, gel filtration chromatography,
reverse phase chromatography, chromatofocusing and
differential solubilization.
In addition, an MEKK protein of the present invention
can be produced by isolating the MEKK protein from cells
expressing the MEKK protein recovered from an animal. For
example, a cell type, such as T cells, can be isolated from
the thymus of an animal. MEKK protein can then be isolated
from the isolated T cells using standard techniques
described herein.
The present invention also includes a method to
identify compounds capable of regulating signals initiated
from a receptor on the surface of a cell, such signal
regulation involving in some respect, MEKK protein. Such
a method comprises the steps of: (a) contacting a cell
containing an MEIaC protein with a putative regulatory
compound; (b) contacting the cell with a ligand capable of
binding to a receptor on the surface of the cell; and (c)
assessing the ability of the putative regulatory compound
to regulate cellular signals by determining activation of
a member of an MEKK-dependent pathway of the present
invention. A preferred method to perform step (c)
comprises measuring the phosphorylation of a member of an
WO 95128421 PCT/L1S94/11690
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MEKK-dependent pathway. Such measurements can be performed
using immunoassays having antibodies specific for
phosphotyrosines, phosphoserines and/or phosphothreonines.
Another preferred method to perform step (c) comprises
measuring the ability of the MEKK protein to phosphorylate
MEK protein and/or JEK protein using methods described
herein.
In another embodiment, a method to identify compounds
capable of regulating signal transduction in a cell can
comprise the steps of: (a) contacting a putative inhibitory
compound with an MEKK protein to form a reaction mixture;
(b) contacting the reaction mixture with MEK protein; and
(c) assessing the ability of the putative inhibitory
compound to inhibit phosphorylation of the MEK protein by
the MEKK protein. The results obtained from step (c) can
be compared with the ability of a putative inhibitory
compound to inhibit the ability of Raf protein to
phosphorylate MEK protein, to determine if the compound can
selectively regulate signal transduction involving MEKK
protein independent of Raf protein. MEKK, MEK and Raf
proteins used in the foregoing methods can be recombinant
proteins or naturally-derived proteins.
Moreover, one can determine whether the site of
inhibitory action along a particular signal transduction
pathway involves both Raf and MEKK proteins by carrying out
experiments set forth above (i.e., see discussion on MEKK-
dependent pathways).
~'1 1
WO 95/28421 ~ ~ ~ ~ PCT/US94/11690
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Another aspect of the present invention includes a kit
to identify compounds capable of regulating signals
initiated from a receptor on the surface of a cell, such
signals involving in some respect, MEKK protein. Such kits
include: (a) at least one cell containing MEKK protein; (b)
a ligand capable of binding to a receptor on the surface of
the cell; and (c) a means for assessing the ability of a
putative regulatory compound to alter phosphorylation of
the MEKK protein. Such a means for detecting
phosphorylation include methods and reagents known to those
of skill in the art, for example, phosphorylation can be
detected using antibodies specific for phosphorylated amino
acid residues, such as tyrosine, serine and threonine.
Using such a kit, one is capable of determining, with a
fair degree of specificity, the location along a signal
transduction pathway of particular pathway constituents, as
well as the identity of the constituents involved in such
pathway, at or near the site of regulation.
In another embodiment, a kit of the present invention
can includes: (a) MEKK protein; (b) MEK protein; and (c) a
means for assessing the ability of a putative inhibitory
compound to inhibit phosphorylation of the MEK protein by
the MEKK protein. A kit of the present invention can
further comprise Raf protein and a means for detecting the
ability of a putative inhibitory compound to inhibit the
ability of Raf protein to phosphorylate the MEK protein.
Another aspect of the present invention relates to the
treatment of an animal having a medical disorder that is
WO 95/28421 ~ PCT/US94111690
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subject to regulation or cure by manipulating a signal
transduction pathway in a cell involved in the disorder.
Such medical disorders include disorders which result from
abnormal cellular growth or abnormal production of secreted
cellular products. In particular, such medical disorders
include, but are not limited to, cancer, autoimmune
disease, inflammatory responses, allergic responses and
neuronal disorders, such as Parkinson's disease and
Alzheimer's disease. Preferred cancers subject to
treatment using a method of the present invention include,
but are not limited to, small cell carcinomas, non-small
cell lung carcinomas with overexpressed EGF receptors,
breast cancers with overexpressed EGF or Neu receptors,
tumors having overexpressed growth factor receptors of
established autocrine loops and tumors having overexpressed
growth factor receptors of established paracrine loops.
According to the present invention, the term treatment can
refer to the regulation of the progression of a medical
disorder or the complete removal of a medical disorder
(e. g., cure). Treatment of a medical disorder can comprise
regulating the signal transduction activity of a cell in
such a manner that a cell involved in the medical disorder
no longer responds to extracellular stimuli (e. g., growth
factors or cytokines), or the killing of a cell involved in
the medical disorder through cellular apoptosis.
One aspect of the present invention involves the
recognition that an MEKK protein of the present invention
is capable of regulating the homeostasis of a cell by
a WO 95/28421 ~ i ~ ~ ~ ~ PCT/US94/11690
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regulating cellular activity such as cell growth cell
death, and cell function (e. g., secretion of cellular
products). Such regulation, in most cases, is independent
of Raf, however, as discussed above (and as shown in Fig.
2), some pathways capable of regulation by MEKK protein may
be subject to upstream regulation by Raf protein.
Therefore, it is within the scope of the present invention
to either stimulate or inhibit the activity of Raf protein
and/or MEKK protein to achieve desired regulatory results.
Without being bound by theory, it is believed that the
regulation of Raf protein and MEKK protein activity at the
divergence point from Ras protein (see Fig. 2) can be
controlled by a "2-hit" mechanism. For example, a first
"hit" can comprise any means of stimulating Ras protein,
thereby stimulating a Ras-dependent pathway, including, for
example, contacting a cell with a growth factor which is
capable of binding to a cell surface receptor in such a
manner that Ras protein is activated. Following activation
of Ras protein, a second "hit" can be delivered that is
capable of increasing the activity of JNK activity compared
with MAPK activity, or vice versa. A second "hit" can
include, but is not limited to, regulation of JNK or MAPK
activity by compounds capable of stimulating or inhibiting
the activity of MEKK, JEK, Raf and/or MEK. For example,
compounds such as protein kinase C or phospholipase C
kinase, can provide the second "hit" needed to drive the
divergent Ras-dependent pathway down the MEKK-dependent
218~~?6
WO 95/28421 PCT/US94/11690
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pathway in such a manner that JNK is preferentially
activated over MAPK.
One embodiment of the present invention comprises a
method for regulating the homeostasis of a cell comprising
regulating the activity of an MEKK-dependent pathway
relative to the activity of a Raf-dependent pathway in the
cell. As used herein, the term "homeostasis" refers to the
tendency of a cell to maintain a normal state using
intracellular systems such as signal transduction pathways.
Regulation of the activity of an MEKK-dependent pathway
includes increasing the activity of an MEKK-dependent
pathway relative to the activity of a Raf-dependent pathway
by regulating the activity of a member of an MEKK-dependent
pathway, a member of a Raf-dependent pathway, and
combinations thereof, to achieve desired regulation of
phosphorylation along a given pathway, and thus effect
apoptosis. Preferred regulated members of an MEKK-
dependent pathway or a Raf-dependent pathway to regulate
include, but are not limited to, proteins including MEKK,
Raf, JEK, MEK, MAPK, JNK, TCF, ATF-2, Jun and Myc, and
combinations thereof.
In one embodiment, the activity of a member of an
MEKK-dependent pathway, a member of a Raf-dependent
pathway, and combinations thereof, are regulated by
altering the concentration of such members in a cell. One
preferred regulation scheme involves altering the
concentration of proteins including MEKK, Raf, JEK, MEK,
MAPK, JNK, TCF, Jun, ATF-2, and Myc, and combinations
~ ~'~526
WO 95/28421 PCT/US94111690
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thereof. A more preferred regulation scheme involves
increasing the concentration of proteins including MEKK,
JEK, JNK, Jun, ATF-2, and Myc, and combinations thereof.
Another more preferred regulation scheme involves
decreasing the concentration of proteins including Raf,
MEK, MAPK, and TCF, and combinations thereof. It is also
within the scope of the present invention that the
regulation of protein concentrations in two or more of the
foregoing regulation schemes can be combined to achieve an
optimal apoptotic effect in a cell.
A preferred method for increasing the concentration of
a protein in a regulation scheme of the present invention
includes, but is not limited to, increasing the copy number
of a nucleic acid sequence encoding such protein within a
cell, improving the efficiency with which the nucleic acid
sequence encoding such protein is transcribed within a
cell, improving the efficiency with which a transcript is
translated into such a protein, improving the efficiency of
post-translational modification of such protein, contacting
cells capable of producing such protein with anti-sense
nucleic acid sequences, and combinations thereof.
In a preferred embodiment of the present invention,
the homeostasis of a cell is controlled by regulating the
apoptosis of a cell. A suitable method for regulating the
apoptosis of a cell is to regulate the activity of an MEKK-
dependent pathway in which the MEKK protein regulates the
pathway substantially independent of Raf. A particularly
preferred method for regulating the apoptosis of a cell
21 ~;05~~
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comprises increasing the concentration of MEKK protein by
contacting a cell with a nucleic acid molecule encoding an
MEKK protein that possesses unregulated kinase activity.
A preferred nucleic acid molecule with which to contact a
cell includes a nucleic acid molecule encoding an MEKK
protein represented by SEQ ID N0:2, SEQ ID N0:4, SEQ ID
N0:6, SEQ ID N0:8 and SEQ ID NO:10, and combinations
thereof . A more preferred nucleic acid molecule with which
to contact a cell includes a nucleic acid molecule encoding
l0 a truncated MEKK protein having only the kinase catalytic
domain (i.e., no regulatory domain) of an MEKK protein
represented by SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ
ID N0:8 and/or SEQ ID NO:10. An even more preferred
nucleic acid molecule with which to contact a cell includes
a nucleic acid molecule including MEKKl , MEKK2 ,
352-672 352-619
MEKK335s-626 MEKK4811_1195, MEKK5~_1247, and combinations thereof .
Again, suitable variations of MEKK proteins described
herein comprise those proteins encoded by a nucleic acid
molecule that are able to hybridize to any of the above
sequences under stringent conditions.
It is within the scope of the invention that the
foregoing method can further comprise the step of
decreasing the activity of MEK protein in the cell by
contacting the cell with a compound capable of inhibiting
MEK activity. Such compounds can include: peptides capable
of binding to the kinase domain of MEK in such a manner
that phosphorylation of MAPK protein by the MEK protein is
inhibited; and/or peptides capable of binding to a portion
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of a MAPK protein in such a manner that phosphorylation of
the MAPK protein is inhibited.
In another embodiment, the activity of a member of an
MEKK-dependent pathway, a member of a Raf-dependent
pathway, and combinations thereof, can be regulated by
directly altering the activity of such members in a cell.
A preferred method for altering the activity of a member of
an MEKK-dependent pathway, includes, but is not limited to,
contacting a cell with a compound capable of directly
interacting with a protein including MEKK, JEK, JNK, Jun,
ATF-2, and Myc, and combinations thereof, in such a manner
that the proteins are activated; and/or contacting a cell
with a compound capable of directly interacting with a
protein including Raf, MEK, MAPK, TCF protein, and
combinations thereof in such a manner that the activity of
the proteins are inhibited. A preferred compound with
which to contact a cell that is capable of regulating a
member of an MEKK-dependent pathway includes a peptide
capable of binding to the regulatory domain of proteins
including MEKK, JEK, JNK, Jun, ATF-2, and Myc, in which the
peptide inhibits the ability of the regulatory domain to
regulate the activity of the kinase domains of such
proteins. Another preferred compound with which to contact
a cell includes TNFa, growth factors regulating tyrosine
kinases, hormones regulating G protein-coupled receptors
and FAS ligand.
A preferred compound with which to contact a cell that
is capable of regulating a member of a Raf-dependent
i i
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pathway includes a peptide capable of binding to the kinase
catalytic domain of a protein selected from the group
consisting of Raf, MEK-1, MEK-2, MAPK, and TCF, in which
the peptide inhibits the ability of the protein to be
phosphorylated or to phosphorylate a substrate.
One aspect of the present invention relates to the
recognition that an MEKK protein is capable of activating
MAPK. MAPK is known to be involved in various cellular
pathways in mammalian systems. MAPK is known to be
involved in cellular mitogenesis, DNA synthesis, cell
division and differentiation. MAPK is also recognized as
being involved in the activation of oncogenes, such as c-
jun and c-myc. While not bound by theory, the present
inventor believes that MAPK is also intimately involved in
various abnormalities having a genetic origin. MAPK is
known to cross the nuclear membrane and is believed to be
at least partially responsible for regulating the
expression of various genes. As such, MAPK is believed to
play a significant role in the instigation or progression
of cancer, neuronal diseases, autoimmune diseases, allergic
reactions, wound healing and inflammatory responses. The
present inventor, by being first to identify nucleic acid
sequences encoding MEKK, recognized that it is now possible
to regulate the expression of MEKK, and thus regulate the
activation of MAPK.
The present invention also includes a method for
regulating the homeostasis of a cell comprising injecting
an area of a subject's body with an effective amount of a
WO 95/28421 ~1 6 5 2 6 pCT~S94/11690
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naked plasmid DNA compound. A naked plasmid DNA compound
comprises a nucleic acid molecule encoding an MEKK protein
of the present invention, operatively linked to a naked
plasmid DNA vector capable of being taken up by and
expressed in a recipient cell located in the body area. A
preferred naked plasmid DNA compound of the present
invention comprises a nucleic acid molecule encoding a
truncated MEKK protein having deregulated kinase activity.
Preferred naked plasmid DNA vectors of the present
invention include those known in the art. When
administered to a subject, a naked plasmid DNA compound of
the present invention transforms cells within the subject
and directs the production of at least a portion of an MEKK
protein or RNA nucleic acid molecule that is capable of
regulating the apoptosis of the cell.
A naked plasmid DNA compound of the present invention
is capable of treating a subject suffering from a medical
disorder including cancer, autoimmune disease, inflammatory
responses, allergic responses and neuronal disorders, such
as Parkinson's disease and Alzheimer's disease. For
example, a naked plasmid DNA compound can be administered
as an anti-tumor therapy by injecting an effective amount
of the plasmid directly into a tumor so that the plasmid is
taken up and expressed by a tumor cell, thereby killing the
tumor cell. As used herein, an effective amount of a
naked plasmid DNA to administer to a subject comprises an
amount needed to regulate or cure a medical disorder the
naked plasmid DNA is intended to treat, such mode of
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administration, number of doses and frequency of dose
capable of being decided upon, in any given situation, by
one of skill in the art without resorting to undue
experimentation.
Therapeutic compounds for use with a treatment method
of the present invention can further comprise suitable
excipients. A therapeutic compound for use with a
treatment method of the present invention can be formulated
in an excipient that the subject to be treated can
tolerate. Examples of such excipients include water,
saline, Ringer's solution, dextrose solution, Hank's
solution, and other aqueous physiologically balanced salt
solutions. Nonaqueous vehicles, such as fixed oils, sesame
oil, ethyl oleate, or triglycerides may also be used.
Other useful excipients include suspensions containing
viscosity enhancing agents, such as sodium
carboxymethylcellulose, sorbitol, or dextran. Excipients
can also contain minor amounts of additives, such as
substances that enhance isotonicity and chemical stability.
Examples of buffers include phosphate buffer, bicarbonate
buffer and Tris buffer, while examples of preservatives
include thimerosal, m- or o-cresol, formalin and benzyl
alcohol. Standard formulations can either be liquid
injectables or solids which can be taken up in a suitable
liquid as a suspension or solution for injection. Thus, in
a non-liquid formulation, the excipient can comprise
dextrose, human serum albumin, preservatives, etc., to
WO 95/28421 2 ~ ~ ~ C) ~ ~ PCT/US94/11690
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which sterile water or saline can be added prior to
administration.
In another embodiment, a therapeutic compound for use
with a treatment method of the present invention can also
comprise a carrier. Carriers are typically compounds that
increase the half-life of a therapeutic compound in the
treated animal. Suitable carriers include, but are not
limited to, liposomes, micelles, cells, polymeric
controlled release formulations, biodegradable implants,
bacteria, viruses, oils, esters, and glycols. Preferred
carriers include liposomes and micelles.
A therapeutic compound for use with a treatment method
of the present invention can be administered to any subject
having a medical disorder as herein described. Acceptable
protocols by which to administer therapeutic compounds of
the present invention in an effective manner can vary
according to individual dose size, number of doses,
frequency of dose administration, and mode of
administration. Determination of such protocols can be
accomplished by those skilled in the art without resorting
to undue experimentation. An effective dose refers to a
dose capable of treating a subject for a medical disorder
as described herein. Effective doses can vary depending
upon, for example, the therapeutic compound used, the
medical disorder being treated, and the size and type of
the recipient animal. Effective doses to treat a subject
include doses administered over time that are capable of
regulating the activity, including growth, of cells
PCT/US94/11690
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involved in a medical disorder. For example, a first dose
of a naked plasmid DNA compound of the present invention
can comprise an amount of that causes a tumor to decrease
in size by about 10% over 7 days when administered to a
subject having a tumor. A second dose can comprise at
least the same the same therapeutic compound than the first
dose.
Another aspect of the present invention includes a
method for prescribing treatment for subjects having a
medical disorder as described herein. A preferred method
for prescribing treatment comprises: (a) measuring the MEKK
protein activity in a cell involved in the medical disorder
to determine if the cell is susceptible to treatment using
a method of the present invention; and (b) prescribing
treatment comprising regulating the activity of an MEKK-
dependent pathway relative to the activity of a Raf-
dependent pathway in the cell to induce the apoptosis of
the cell. The step of measuring MEKK protein activity can
comprise: (1) removing a sample of cells from a subject;
(2) stimulating the cells with a TNFa; and (3) detecting
the state of phosphorylation of JEK protein using an
immunoassay using antibodies specific for phosphothreonine
and/or phosphoserine.
The present invention also includes antibodies capable
of selectively binding to an MEKK protein of the present
invention. Such an antibody is herein referred to as an
anti-MEKK antibody. Polyclonal populations of anti-MEKK
antibodies can be contained in an MEKK antiserum. MEKK
WO 95/28421 _ 2 ~ ~~ y ~~ ~ ~ ~ PCT/US94/11690
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antiserum can refer to affinity purified polyclonal
antibodies, ammonium sulfate cut antiserum or whole
antiserum. As used herein, the term "selectively binds to"
refers to the ability of such an antibody to preferentially
bind to MEKK proteins. Binding can be measured using a
variety of methods known to those skilled in the art
including immunoblot assays, immunoprecipitation assays,
enzyme immunoassays (e. g., ELISA), radioimmunoassays,
immunofluorescent antibody assays and immunoelectron
microscopy; see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press, 1989.
Antibodies of the present invention can be either
polyclonal or monoclonal antibodies and can be prepared
using techniques standard in the art. Antibodies of the
present invention include functional equivalents such as
antibody fragments and genetically-engineered antibodies,
including single chain antibodies, that are capable of
selectively binding to at least one of the epitopes of the
protein used to obtain the antibodies. Preferably,
antibodies are raised in response to proteins that are
encoded, at least in part, by a MEKK nucleic acid molecule.
More preferably antibodies are raised in response to at
least a portion of an MEKK protein, and even more
preferably antibodies are raised in response to either the
amino terminus or the carboxyl terminus of an MEKK protein.
Preferably, an antibody of the present invention has a
WO 95/28421 ~ i ~ PCT/US94/11690
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single site binding affinity of from about 103M'~ to about
lO~zM-~ for an MEKK protein of the present invention.
A preferred method to produce antibodies of the
present invention includes administering to an animal an
effective amount of an MEKK protein to produce the antibody
and recovering the antibodies. Antibodies of the present
invention have a variety of potential uses that are within
the scope of the present invention. For example, such
antibodies can be used to identify unique MEKK proteins and
recover MEKK proteins.
Another aspect of the present invention comprises a
therapeutic compound capable of regulating the activity of
an MEKK-dependent pathway in a cell identified by a
process, comprising: (a) contacting a cell with a putative
regulatory molecule; and (b) determining the ability of the
putative regulatory compound to regulate the activity of an
MEKK-dependent pathway in the cell by measuring the
activation of at least one member of said MEKK-dependent
pathway. Preferred methods to measure the activation of a
member of an MEKK-dependent pathway include measuring the
transcription regulation activity of c-Myc protein,
measuring the phosphorylation of a protein selected from
the group consisting of MEKK, JEK, JNK, Jun, ATF-2, Myc,
and combinations thereof.
The following examples are provided for the purposes
of illustration and are not intended to limit the scope of
the~present invention.
EXAMPLES
WO 95128421 PCT/US94/11690
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Example 1
This example describes the structural characterization
of MEKK 1 protein.
A. MEKK 1 Nucleotide Sequence
MEKK 1 protein was cloned by the following method.
Unique degenerate inosine oligodeoxynucleotides were
designed to correspond to regions of sequence identity
between the yeast Stell and Byr2 genes. With primers and
cDNA templates derived from polyadenylated RNA from NIH 3T3
cells, a polymerase chain reaction (PCR) amplification
product of 320 base pairs (bp) was isolated. This 320 by
cDNA was used as a probe to identify an MEKK 1 cDNA of 3260
by from a mouse brain cDNA library using standard methods
in the art. The MEKK 1 nucleotide sequence was determined
by dideoxynucleotide sequencing of double-stranded DNA
using standard methods in the art.
Referring to Table 6, based on the Kozak consensus
sequence for initiation codons, the starting methionine can
be predicted to occur at nucleotide 486. With this
methionine at the start, the cDNA encodes a protein of 672
amino acids, corresponding to a molecular size of 73 kD.
There is another in-frame methionine at position 441, which
does not follow the Kozak rule, but would yield a protein
of 687 amino acid residues (74.6 kD). Also referring to
Table 6, 20% of the NHZ-terminal 400 amino acids are serine
or threonine and there are only two tyrosines. Several
potential sites of phosphorylation by protein kinase C are
WO 95/28421 , i ~ ~ PCT/US94/11690
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apparent in the NH2-terminal region. The kinase catalytic
domain is located in the COOH-terminal half of the MEKK 1.
B. Southern Blot Analysis of MEKK 1 Transcr ~t
Equal amounts (20 fig) of total RNA were loaded onto
the gel as indicated by ethidium bromide staining. Blots
were probed with either a 320-by cDNA fragment encoding a
portion of the MEKK kinase domain or an 858-by fragment
encoding a portion of the NHZ terminal region of MEKK using
standard methods in the art. Referring to Fig. 3A, a 7.8
kb mRNA was identified with probes derived from both the 5'
and 3' ends of the MEKK cDNA in several cell lines and
mouse tissues. The MEKK mRNA was highly expressed in mouse
heart and spleen, an in lower amounts in liver.
C. Southern Blot Analysis
Mouse genomic DNA (10 fig) was digested with either Bam
HI, Hind III or Eco RI and applied to gels using standard
methods in the art. Blots were probed with a 320-by
fragment of the MERR gene. Fig. 3B shows the appearance of
one band in the Bam HI and Hind III digests which indicates
that MEKK is encoded by one gene. The appearance of two
bands in the Eco RI digest indicates the likely presence of
an Eco RI site within an intron sequence spanned by the
probe.
D. Immunoblots Using Anti-MEKK Antibodies
Three polyclonal antisera were prepared using three
different antigens. A first polyclonal antiserum was
prepared using an antigen comprising a 15 amino acid
peptide DRPPSRELLKHPVER derived from the COOH-terminus of
rv WO 95/28421 C I ~ b J PCT/LTS94/11690
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MEKK. NZW rabbits were immunized with the peptide and
antisera was recovered using standard methods known in the
art. This first polyclonal antiserum is hereinafter
referred to as the DRPP antiserum.
A second polyclonal antiserum was produced using a DNA
clone comprising an MERIC cDNA digested with EcoRl and PstI,
thereby creating a 1270 by fragment that encodes the amino
terminus of MEKK. This fragment was cloned into pRSETC to
form the recombinant molecule pMEKK~_~9 comprising amino
acid residues 1 to 369 of MEKK 1. The pMEKKl~_369
recombinant molecule was expressed in E. coli and protein
encoded by the recombinant molecule was recovered and
purified using standard methods known in the art. NZW
rabbits were immunized with the purified recombinant MEKK1~-
~9 protein and antisera was recovered using standard methods
known in the art. This second polyclonal antiserum is
hereinafter referred to as the MEKK1~_~9 antiserum.
A third polyclonal antiserum was produced using a DNA
clone comprising an MEICIC cDNA digested with Pst 1 and Rpn
Z, thereby creating a 1670 by fragment that encodes the
catalytic domain of MEKK. This fragment was cloned into
pRSETC to form the recombinant molecule pMEKK3~o-r38
comprising amino acid residues 370 to 738 of MEKK 1
(encoded by base pairs 1592-3260) . The pMEKKl3~o-738
recombinant molecule was expressed in E. coli and protein
encoded by the recombinant molecule was recovered and
purified using standard methods known in the art. NZW
rabbits were immunized with the purified recombinant
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MEKKI3~o_~$ protein and antisera was recovered using standard
methods known in the art. This second polyclonal antiserum
is hereinafter referred to as the MEKKI3~o-r3a antiserum.
The DRPP antiserum was used to probe Western Blots of
soluble cellular protein derived from several rodent cell
lines. Soluble cellular protein (100 ~u,g) or recombinant
MEKK COOH-terminal fusion protein (30 ng) was loaded onto
a 10% Tris Glycine SDS-PAGE gel and the protein transferred
to a nylon filter using methods standard in the art. The
l0 nylon filter was immunoblotted with affinity purified DRPP
antiserum (1:300 dilution). Referring to Fig. 3C, a 78 kD
immunoreactive protein was identified in the samples
comprising protein from Pheochromocytoma (PC12), Rat la,
and NIH 3T3 cells. A prominent 50 kD immunoreactive band
was also commonly present but varied in intensity from
preparation to preparation indicating the band is a
proteolytic fragment. Visualization of both the 78 kD and
50 kD immunoreactive bands on immunoblots was inhibited by
pre-incubation of the 15 amino acid peptide antigen with
the affinity purified DRPP antiserum. The MEKK protein
detected by immunoblotting is similar to the molecular size
predicted from the open reading frame of the MERR cDNA.
In a second immunoblot experiment, PC12 cells
stimulated or not stimulated with EGF were lysed and
resolved on 10% Tris Glycine SDS-PAGE gel as described
above. MEKK proteins contained in the cell lysates were
identified by immunoblot using affinity purified MEKKl~_369
antiserum (1:300) using methods standard in the art.
WO 95/28421 ~ ~ PCT/US94/11690
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Referring to Fig. 4, MEKK 1 and two higher molecular weight
proteins having MEKK activity, MEKK a and MEKK B, were
identified using the affinity purified MEKK1~_369 antiserum.
MEKK 1, and not MEKK a and MEKK B, were identified using
the affinity purified MEKKl~_~9 antiserum.
Using the same procedure described above, two MEKK
immunoreactive species of approximately 98 kD and 82 kD
present in PC12, Ratla, NIH3T3, and Swiss3T3 cell lysates
were recognized by affinity purified MEKK1~_369 antiserum as
shown in Fig. 5. It should be noted that the 98 kD MEKK
protein described herein was originally identified as a 95
kD MEKK protein in the related PCT application
(International application no. PCT/US94/04178). Subsequent
Tris Glycine SDS-PAGE gel analysis has led to the
determination that the modification in molecular weight.
Visualization of both of these proteins was inhibited by
incubation of the affinity purified MEKKi~_~9 antiserum with
purified recombinant MEKK1~_~9 fusion protein antigen. A
single 98' kD MEKIt protein was present in MEKK
immunoprecipitates, but not in immunoprecipitates using
preimmune serum. More of the 98 kD MEKK was expressed in
PC12 cells relative to fibroblast cell lines.
Immunoblotting with antibodies that specifically recognize
Raf-1 or Raf-B indicated that neither of these enzymes were
present as contaminants of MEKK immunoprecipitates. 98 kD
MEKK in MEKK immunoprecipitates did not comigrate with Raf-
1 or~ Raf-B in PC12 cell lysates and no cross-reactivity
between MEKK and Raf antibodies was observed.
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Examgle 2
This example describes the isolation of nucleic acid
sequences encoding MEI~ 2, MEKR 3 and MEKK 4 protein.
PCR primers were designed based on the nucleotide
sequence of MEKIC 1. PCR amplification of fragments from
DNA isolated from reverse transcriptase reactions of RNA
isolated form PC12 and HL60 cells was conducted using
standard techniques. The resultant PCR products were cloned
into the pGEX cloning vector (Promega, Wisconsin) using
l0 standard procedures and submitted to DNA sequence analysis
using standard techniques.
Example 3
This example describes the expression of MEKK 1
protein in COS-1 cells to define its function in regulating
the signaling system that includes MAPK.
COS cells in 100-mm culture dishes were transfected
with either the pCVMVS expression vector alone (1 ~Cg:
control) or the pCVMVS MEKK construct (1 /gig: MEKK). After
48 hours, the cells were placed in serum-free medium
containing bovine serum albumin (0.1 percent) f or 16 to 18
hours to induce quiescence. Cells were then treated with
human EGF (30 ng/ml)(+EGF) or buffer (control) for to
minutes, washed twice in cold phosphate buffered saline
(PBS), and lysed in cell lysis buffer containing 50 mM B-
glycerophosphate (pH 7.2), 100 y~M sodium vanadate, 2 mM
MgCl2, 1mM EGTA Triton X-100*(0.5 percent), leupeptin {2
~cg/ml) , aprotinin (2~Cg/ml) , and 1 mM dithiothreitol (600
~cl). After centrifugation for l0 minutes at maximum speed
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WO 95128421 PCTlUS94111690
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in a microfuge, COS cell lysates containing 0.5 to 1 mg of
* ~.
soluble protein were subjected to FPLC on a MONO Q column,
and eluted fractions were assayed f or MAPK activity
according to the method described in Heasley et al., p.
545, 1992, Mol. Biol. Cell, Vol. 3.
Referring to Fig. 6A, when MEKK 1 was overexpressed in
COS 1 cells, MAPK activity was four to five times greater
than that in control cells transfected with plasmid lacking
an MEKK 1 cDNA insert. The activation of MAPK occurred in
COS cells deprived of serum and in the absence of any added
growth factor. The activity of MAPK was similar to that
observed after stimulation of control cells with EGF.
Stimulation of CoS cells transiently overexpressing ME~C
with EGF resulted in only a slight increase in MAPK
activity compared to that observed with MEKK expression
alone.
To ensure that ME'fQC protein was present in the samples
tested for MAPK activity, protein from cell lysates of the
transfected COS 1 cells were immunoblotted with MEKK
specific antiserum. Egual amounts (100 fig) of soluble
protein lysate from COS cells were placed on the gel for
immunoblotting using the methods described in Example 1.
The filters were immunoblotted using the affinity purified
DRPP antiserum (1:300) and affinity purified M~1.369
antiserum (1:300). Referring to Fig. 6B, the results E,.;
indicate that expression of MEKK in cells transfected with
vector encoding MEN that were treated with or Without EGF.
Only the 50 kD MEKK immunoreactive fragment was detected in
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WO 95/28421 ~ ~ ~ b ~ ~ 6 PCT/US94/11690
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lysates from control COS cells using the DRPP antiserum.
Transient expression of MEKK in COS cells yielded a
predominant 82 kD band that was slightly larger than that
observed in PC12 , Rat la, or NIH 3T3 cells . Addition of
the 15 amino acid DRPP peptide antigen to the antiserum
during immunoblotting prevented detection of all of the
immunoreactive bands; these bands were not detected in
extracts of control COS cells, an indication that they were
derived from the expressed MEKK protein.
Example 4
This Example describes the expression of MEKK 1 in COS
cells to test the ability of MEKK protein to activate MEK
protein.
Recombinant MAPK was used to assay MEK activity in COS
cell lysates that had been fractionated by fast protein
liquid chromatography (FPLC) on a Mono S column. A cDNA
encoding p42 MAPK from Xenopus laevis was cloned into the
pRSETB expression vector. This construct was used for
expression in the LysS strain of Escherichia coli BL21(DE3)
of a p42 MAPK fusion protein containing a polyhistidine
sequence at the NH2-terminus. Cultures containing the
expression plasmid were grown at 37°C to an optical density
of 0.7 to 0.9 at 600 nM. Isopropyl-B-thiogalactopyranoside
(0.5 mM) was added to induce fusion protein synthesis and
the cultures were incubated for 3 hours. The cells were
then collected and lysed by freezing, thawing, and
sonication. The lysate was centrifuged at 10,0008 for 15
minutes at 4°C. The supernatant was then passed over a Ni2+-
WO 95/28421 21 g 6 5 2 6 v~ p~~S94111690
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charged Sepharose resin and the soluble recombinant MAPK
was eluted in sodium phosphate buffer (pH 4.5). The
purified recombinant MAPK was more than 80 percent pure.
The purified recombinant MAPK served as a substrate for MEK
and catalyzed the phosphorylation of a peptide consisting
of residues 662 to 681 of the EGF receptor (EGFR~2~~~).
Soluble cell lysates from COS cells transiently
transfected with MEKR, mock-transfected (control) , or mock-
transfected and treated with EGF (30 ng/ml) (~EGF), were
fractionated by FPLC on a Mono S column and endogenous MEK
activity was measured. Endogenous MAPK eluted in fractions ,
2 to 4, whereas MEK was contained in fractions 9 to 13.
For assaying endogenous MEK activity, cells were washed
twice in cold PBS and lysed in 650 ~cl of a solution
containing 50 mM B-glycerophosphate, 10 mM z-rr-
morpholinoethane-sulfonic acid (pH 6.0), 100 ~M sodium
vanadate, 2 mM MgClz, 1 mM EGTA, Triton X-100 (0.5 percent),
leupeptin (5 ~g/ml), aprotinin (2 ~.g/ml), and 1 mM
dithiothreitol. After centrifugation at maximum speed for
10 minutes in a microfuge, soluble cell lysates (1 to 2 mg
of protein) were applied to a Mono S~column equilibrated in
elution buffer (50 mM B-glycerophosphate, 10 mM MES (pH
6.0), 100 ACM sodium vanadate, 2 mM MgClz, 1 mM EGTA, and 1
mM dithiothreitol. The column was washed with buffer (2
ml) and bound proteins were eluted with a 30m1 linear
gradient of 0 to 350 mM NaCl in elution buffer. A portion
(30 y~l) of each fraction was assayed for MEK activity by
mixing with buffer (25 mM B-glycerophosphate, 40 mM N-(2-
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WO 95/28421 PCT/US94/11690
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hydroxyethyl)piperazine-N'-(2-ethanolsulfonic acid)(pH7.2)
50 mM sodium vanadate, 10 mM MgClz, 100 ~,M Y-3zP-ATP (3000
to 4000 cpm/pmol), inhibitor protein-20 (IP-20;
TTYADFIASGRTGRRNAIHD; 25 ~g/ml), 0.5 mM EGTA, recombinant
MAP kinase (7.5 ~g/ml) , and 200 ~M EGFR~z-~~) in a final
volume of 40 ~C1. After incubation at 30°C for 20 minutes,
the incorporation of y-3zP-ATP into EGFR~z-~~ was measured.
In this assay, the ability of each column fraction to
activate added recombinant MAPK was measured by the
incorporation of Y-3zP-ATP into the MAPK substrate, a
peptide derived from the EGF receptor (EGFR).
Referring to Fig. 7, the first peak of activity eluted
represents endogenous activated MAPK, which directly
phosphorylates the EGFR peptide substrate. The second peak
of activity represents the endogenous MEK in COS cells.
The activity of endogenous MEK activity was
characterized by fractionation of Mono S FPLC. COS cell
lysates were fractionated by FPLC on a Mono Q column to
partially purify the expressed MEKK. Purified recombinant
MEK-1 was then used as a substrate for MEKK in the presence
of y 3zP-ATP to determine whether MEKK directly
phosphorylates MEK-1.
A cDNA encoding MEK-1 was obtained from mouse B cell
cDNA templates with the polymerase chain reaction and
oligodeoxynucleotide primers corresponding to portions of
the 5' coding region and 3' untranslated region of MEK-1.
The catalytically inactive MEK-1 was generated by site-
directed mutagenesis of Lys~3 to Met. The wild-type MEK-1
WO 95/28421 ~ ~ r~ . PCT/US94111690
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and catalytically inactive MEK-1 proteins were expressed in
pRSETA as recombinant fusion proteins containing a
polyhistidine sequence at their NHZ-termini.
Lysates from COS cells transfected with MEKK or mock
s transfected (control) were subjected to FPLC on a Mono Q
column as described above. Portions (20 N,1) of fractions
containing MEKK were mixed With buffer containing 50 mM 8
glycerophosphate (pH 7.2), 100 ~,M sodium vanadate, 2 mM
MgClz, imM EGTA, 50 ~u,M ATP, IP-20 (50 fcg/ml) , and 10 Ecl y
3zP-ATP in a reaction volume of 40 ~1 and incubated for 40
minutes in the presence (+) or absence (-) of recombinant,
catalytically inactive MEK-1 (150 ng)(kinase-MEK-1).
Reactions were stopped by the addition of 5 x SDS sample
buf f er ( 10 Ecl ) , 1 x SDS bu f f er contains 2 percent SDS , 5
percent glycerol, 62.5 mM tris-HC1 (pH 6.8), 5 percent B-
mercaptoethanol, and 0.001 percent bromophenol blue. The
samples were boiled for 3 minutes and subjected to SDS-PAGE
and autoradiography.
Referring to Fig. 8A, autophosphorylated recombinant
wild-type MEK-1 (WT MEK-1) comigrated with phosphorylated
catalytically inactive MEK-1. Thus, MEKK was capable of
phosphorylating MEK-1. Corresponding fractions of lysates
from control cells, however, were not able to phosphorylate
MEK-1.
Example 5
This example describes studies showing that the
modified form of MEK-1 that was used in the phosphorylation
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assay of Example 4 did not autophosphorylate as does wild-
type MEK-1.
Phosphorylation of ~catalytically inactive MEK-1 by
MEKK was time dependent (Fig. 8B); MEKK was also
phosphorylated. Fraction 22 from FPLC on a Mono Q column
(20 ~u,l) was incubated with or without recombinant
catalytically inactive MEK-1 (0.15 ~r,g) for the indicated
times. Referring to Fig. 8B, phosphorylation of kinase
MEK-1 and MEKK was visable after 5 minutes and maximal
l0 after about 20 minutes. The time-dependent increase in
MEKK phosphorylation correlated with a decreased mobility
of the MEKK protein during SDS-PAGE. Referring to Fig. 8C,
immunoblotting demonstrated that the MEKK protein co-eluted
(after FPLC on a Mono Q column) with the peak of activity
(fraction 22) that phosphorylated MEK. The slowly
migrating species of MEKK were also detected by
immunoblotting. Thus, expression of MEKK appears to
activate MAPK by activating MEK.
Example 6
This Example describes that the phosphorylation of MEK
by overexpressed MEKK resulted in activation of MEK,
recombinant wild-type MEK-1 and a modified form of MAPK
that is catalytically inactive.
COS cell lysates were separated by Mono Q-FPLC and
fractions containing MEKK were assayed for their ability to
activate added wild-type MEK-1 such that it would
phosphorylate catalytically inactive recombinant MAPK in
the presence of Y-32P-ATP. Lysates from COS cells
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transfected with MEKK or mock-transfected (control) were
fractionated by FPLC on a Mono Q column and portions (20
~1) of fractions containing MEKK were mixed with buffer.
Each fraction was incubated in the presence (+) or absence
(-) of purified recombinant wild-type MEK-1 (150 ng) and in
the presence of purified recombinant, catalytically
inactive (kinase-) MAPK (300 ng). Referring to Fig. 9A,
fractions 20 to 24 from lysates of COS cells transfected
with MEKK activated MEK-1. Thus, MEKK phosphorylated and
activated MEK-1, leading to MAPK phosphorylation.
Example 7
This Example describes studies demonstrating that MEKK
activated MEK directly, and not through the activation of
one or more other kinases contained in the column
fractions.
Overexpressed MEKK was immunoprecipitated from COS
cell lysates with affintiy purified MEKK~_~9 antiserum.
Immunoprecipitated MEKK was resuspended in 10 to 15 ~u,l of
PAN (10 mM piperazine-N, N'-bis-2-ethanesulfonic acid
(Pipes) (pH 7.0), 100 mM NaCl, and aprotinin (20 Er,g/ml) and
incubated with (+) or without (-) catalytically inactive
MEK-1 (150 ng) and 25 ~u,Ci of Y-3zp-ATP in 20 mM pipes (pH
7. 0) , 10 mM MnCl2, and aprotinin (20 ~g/ml) in a final voume
of 2 0 E,c 1 f or 15 minutes 3 0 ° C . Reactions were stopped by
the addition of 5 x SDS sample buffer (5 ~C1). The samples
were boiled for 3 minutes and subjected to SDS-PAGE and
autoradiography.
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Referring to Fig. 9B, MEKK phosphorylated
catalytically inactive MEK-1, which comigrated with wild-
type MEK-1 on SDS-PAGE. Several phosphorylated bands of
overexpressed MEKK were detected in the immunoprecipitates.
These bands probably resulted from autophosphorylation of
MEKK and corresponded to the forms of MEKK identified by
immunoblotting of lysates from COS cells transfected with
MEKK. Immunoprecipitates obtained with pre-immune serum
contained no MEKK and did not phosphorylate MEK-1. Thus,
MEKK appears to directly phosphorylate MEK.
Taken together, the results from Examples 4 through 7
show that MEKK can phosphorylate and activate MEK, which in
turn phosphorylates and activates MAPK.
Example 8
This Example demonstrates that Raf can also
phosphorylate and activate MEK.
COS cells deprived of serum were stimulated with EGF,
and Raf was immunoprecipitated with an antibody to the
COOH-terminus of Raf-1. Cos cells were transiently
transfected with vector alone (control) or with the PCV/M5-
MEKK construct (MEKK). Quiescent control cells were
treated with or without human EGF (30 ng/ml) for 10 minutes
and Raf was immunoprecipitatd from cell lysates with an
antibody to a COOH-terminal peptide from Raf.
Immunoprecipitated Raf was incubated with catalytically
inactive MEK-1 (150 ng) and 25 fc.l of y-32P-ATP. The
immunoprecipitated Raf phosphorylated MEK-1 in the presence
of y-3ZP-ATP (Fig. l0A) . Little or no phosphorylation of
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MEK-1 by Raf was observed in immunoprecipitates of Raf from
COS cells overexpressing ~MEKK. Treatment of COS cells
overexpressing MEKK with EGF resulted in a similar degree
of phosphorylation of MEK-1 by immunoprecipitated Raf (Fig.
lOB). Cells transfected with MEKK and deprived of serum
were treated with EGF, and Raf was immunoprecipitated and
incubated with catalytically inactive MEK-1. Equal amounts
of Raf were immunoprecipitated in each sample as
demonstrated by immunoblotting with antibodies to Raf. The
l0 slowest migrating band represents an immunoprecipitated
phosphoprotein that is unrelated to Raf or MEK-1. The
amount of Raf in the immunoprecipitates from control cells
and cells transfected with MEKK was similar as shown by
subsequent SDS-PAGE and immunoblotting with the antibody to
Raf. Thus, both MEKK and Raf can independently activate
MEK.
Example 9
This Example describes the activation of a 98 kD MEKK
protein isolated from PC12 cells in response to stimulation
of cells containing MEKK protein by growth factors.
PC12 cells were deprived of serum by incubation in
starvation media (DMEM, 0.1% BSA) for 18-20 hours and MEKK
was immunoprecipitated from lysates containing equal
amounts of protein from untreated controls or cells treated
with EGF (30ng/ml) or NGF (100ng/ml) for 5 minutes with the
above-described anti-MEKK antibodies speicific for the NH4-
terminal portion of MEKK. Immunoprecipitated MEKK was
resuspended in 8~1 of PAN (lOmM piperazine-N,N'-bis-2-
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ethanesulfonic acid (Pipes) (pH 7.0), 100mM NaCl, and
aprotinin (20~g/ml)) and incubated with catalytically
inactive MEK-1 (150ng) and 40~Ci of (y-3zP)ATP in universal
kinase buffer (20mM piperazine-N, N'-bis-2-ethanesulfonic
acid (Pipes) (pH 7.0), lOmM MnClz, and aprotinin (20ug/ml))
in a final volume of 20~C1 for 25 minutes at 30°C.
Reactions were stopped by the addition of 2X SDS sample
buffer (201). The samples were boiled for 3 minutes and
subjected to SDS-PAGE and autoradiography. Raf-B was
immunoprecipitated from the same untreated and treated PC12
cell lysates as above with an antiserum to a COOH-terminal
peptide of Raf-B (Santa Cruz Biotechnology, Inc.) and
assayed similarly. Raf-1 was immunoprecipitated with an
antiserum to the 12 COOH-terminal amino acids of Raf-1
(Santa Cruz Biotechnology, Inc.). Epidermal growth factor
(EGF) treatment of serum starved PC12 cells resulted in
increased MEKK activity.
Referring to Fig. 11, the results were obtained by
measuring the phosphorylation of purified MEK-1 (a kinase
inactive form) by immunoprecipitates of MEKK in in vitro
kinase assays. NGF stimulated a slight increase in MEKK
activity compared to control immunoprecipitates from
untreated cells. Stimulation of MEKK activity by NGF and
EGF was similar to Raf-B activation by these agents,
although Raf-B exhibited a high basal activity. Activation
of c-Raf-1 by NGF and EGF was almost negligible in
comparison to that of MEKK or Raf-B.
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A timecourse of MEKK stimulation by EGF was
established by immunoprecipitating MEKK or Raf-B protein
from lysates of PC12 cells treated with EGF (30ng/ml) for
0 , 1, 3 , 5 , 10 , or 2 0 minutes and incubating the protein
with catalytically inactive MEK-1 (150ng) and (Y 3zp)ATP as
described above. Data represent the relative magnitude of
the response for each timepoint as quantitated by
phosphorimager analysis of radioactive gels from a typical
experiment. A timecourse of EGF treatment indicated that
MEKK activation reached maximal levels following 5 minutes
and persisted for at least 30 minutes (Fig. 12). Raf-B
exhibited a similar timecourse; peak activity occurred
within 3-5 minutes following EGF treatment and was
persistent for up to 20 minutes.
To further dissociate EGF-stimulated MEKK activity
from that of Raf-B, Raf-B was immunodepleted from cell
lysates prior to MEKK immunoprecipitation. Raf-B was pre-
cleared from lysates of serum-starved PC12 cells which had
been either treated or not treated with EGF (30ng/ml) for
5 minutes. Raf-B was pre-cleared two times using antisera
to Raf-B or using preimmune IgG antisera as a control. The
pre-cleared supernatant was then immunoprecipitated with
either MEKK or Raf-8 antisera and incubated with
catalytically inactive MEK-1 and (Y 32p)ATP as described in
detail above. EGF-stimulated and unstimulated PC12 cell
lysates were precleared with either IgG or Raf-B antisera
and then subjected to immunoprecipitation with MEKK
antiserum or Raf-B antibodies. The results shown in Fig.
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13 indicate that pre-clearing with Raf-B resulted in a 60%
diminution of Raf-B activity as measured by phosphorimager
analysis of Raf-B in vitro kinase assays. EGF-stimulated
MEKK activity was unaffected by Raf-B depletion, suggesting
that Raf-B is not a component of MEKK immunoprecipitates.
At least 40% of the Raf-B activity is resistant to
preclearing with Raf-B antibodies. Recombinant wild type
MEKK over-expressed in COS cells readily autophosphorylates
on serine and threonine residues and the amino-terminus of
MEKK is highly serine and threonine rich. MEKK contained
in immunoprecipitates of PC12 cells were tested for
selective phosphorylation of purified recombinant MEKK
amino-terminal fusion protein in in vitro kinase assays.
Serum-starved PC12 cells were treated with EGF
(30ng/ml) for 5 minutes and equal amounts of protein from
the same cell lysates were immunoprecipitated with either
MEKK, Raf-B, or preimmune antiserum as a control.
Immunoprecipitates were incubated with purified recombinant
MEKK NH2-terminal fusion protein (400ng) and (Y-3zP)ATP as
described above. The results shown in Fig. 14 indicate
that MEKK immunoprecipitated from lysates of EGF-stimulated
and unstimulated PC12 cells robustly phosphorylated the
inert 50 kD MEKK NHZ-fusion protein, while Raf-B or
preimmune immunoprecipitates from EGF-stimulated or
unstimulated cells did not use the MEKK NFi2-fusion protein
as a substrate. Thus, the EGF-stimulated MEKK activity
contained in MEKK immunoprecipites is not due to
contaminating Raf kinases.
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Example 10
This Example describes MEKK activity in FPLC Mono Q
ion.-exchange column fractions of PC12 cell lysates.
Cell lysates were prepared from EGF-stimulated PC12
cells. Portions (900 ~1) of 1 ml column fractions (1 to
525 mM NaCl gradient) were concentrated by precipitation
with trichloroacetic acid and loaded on gels as described
above. The gels were blotted and then immunoblotted with
MEKK specific antibody. The results are shown in Fig. 15A
l0 indicate that 98 kD MEh~C immunoreactivity eluted in
fractions 10 to 12. The peak of B-Raf immunoreactivity
eluted in fraction 14, whereas Raf-1 was not detected in
the eulates from the column. Portions (30 ~I)~of each
fraction from the PC12 lysates of unstimulated control
cells or EGF-treated cells were assayed as described above
in buffer containing purified recombinant MEK-1 (150 ng) as
a substrate. The results shown in Fig. 15B indicate that
the peak of MEKK activity eluted in fractions l0 to 12 from
EGF-stimulated PC12 cells phosphorylated MEK, whereas
little MEK phosphorylation occurred in fractions from
unstimulated cells.
example 11
This Example describes studies demonstrating that the
phosphorylation of both MEK-1 and the MEHIC NHZ-terminal
fusion protein were due to the activity of the 98 kD PC12
cell MEKK.
Cell lysates prepared from EGF-stimulated and
unstimulated cells were fractionated by FPLC on a Mono-Q
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column to partially purify the endogenous MEKK. Lysates
from unstimulated control PC12 cells or cells treated with
EGF (30ng/ml) for 5 minutes were fractionated by FPLC on a
Mono Q column using a linear gradient of 0 to 525 mM NaCl.
A portion (301) of each even numbered fraction was mixed
with buffer (20mM piperazine-N, N'-bis-2-ethanesulfonic acid
(Pipes) (pH 7.0), lOmM MnCl2, aprotinin (20~Cg/ml), 5omM 8-
glycerophosphate (pH.7.2), 1mM EGTA, IP-20 (50~Cg/ml), 50mM
NaF, and 30~Ci (y 3ZP)ATP) containing purified recombinant
to MEK-1 (150ng) as a substrate in a final volume of 401 and
incubated at 30°C for 25 minutes. Reactions were stopped
by the addition of 2X SDS sample buffer (401), boiled and
subjected to SDS-PAGE and autoradiography. The peak of
MEKK activity eluted in fractions 10-12. Portions (301)
of each even numbered fraction from lysates of EGF-treated
PC12 cells were mixed with buffer as described above except
containing purified recombinant MEKK NHZ-terminal fusion
protein (400ng) as a substrate instead of MEK-1. Purified
recombinant kinase inactive MEK-1 or the MEKK NHz-terminal
fusion protein were then used as substrates in the presence
of (y-32P)ATP to determine whether 98 kD MEKK directly
phosphorylates either substrate. Fractions 10-14 of lysate
from PC12 cells treated with EGF phosphorylated MEK-1 while
little MEK-1 phosphorylation occurred in untreated control
fractions. The MEKK NHS-terminal fusion protein was also
phosphorylated in the same fractions as was MEK-1, although
the peak of activity was slightly broader (fractions 8-16).
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Referring to Fig. 16, immunoblotting of column
fractions demonstrated that the 98 kD MEKK protein co-
eluted with the peak of activity that phosphorylated either
exogenously added kinase inactive MEK-1 or the 50 kD MEKK
NHZ-terminal fusion protein. Portions (9001) of even
numbered column fractions were concentrated by
precipitation with trichloroacetic acid and immunoblotted
with MEKK antibody. The peak of immunoreactivity eluted in
fractions 10-12.
Example 12
This Example describes the activation of MEK by a 98
kD MEKK.
98 kD MEKK was immunoprecipitated using the MEKK~_369
antiserum described in Example 1 from untreated (-) or EGF
treated (+) PC12 cell lysates. The immunoprecipitates were
incubated in the presence (+) or absence (-) of purified
recombinant wild-type MEK (150 ng) and in the presence of
purified recombinant catalytically inactive MAPK (300 ng)
and (Y-32P)ATP. The results shown in Fig. 17A indicate that
immunoprecipitated MEKK from EGF-stimulated cells
phosphorylated and activated MEK, leading to MAPK
phosphorylation. No phosphorylation of MAPK occurred in
the absence of added recombinant MEK. Immunoblotting
demonstrated that there was no contaminating MAPK (Fig.
17B) or contaminating MEK (Fig. 17C) in the MEKK
immunoprecipitates from the EGF-stimulated PC12 cells.
Thus, phosphorylation and activation of MEK is due to EGF
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stimulation of MEKK activity measured in the
immunoprecipitates.
Example 13
This Example describes whether 98 kD PC12 cell MEKK
and Raf-B require functional Ras proteins for growth factor
mediated signalling.
Dominant negative Ha-ras(Asn 17) (N~TRas) was expressed
in PC12 cells and EGF-stimulated MEKK or Raf-B activation
was assayed in immunoprecipitates using kinase inactive
MEK-1 as a substrate. PC12 cells stably expressing
dexamethasone inducible N~~Ras were serum starved for 18-20
hours in media containing 0.1% BSA with or without 1~,M
dexamethasone and then untreated or treated with EGF
(30ng/ml) for 5 minutes. Equal amounts of soluble protein
from cell lysates was immunoprecipitated with either MEKK
or Raf-B antisera and incubated with purified recombinant
catalytically inactive MEK-1 and (y 32P)ATP as described
above. Expression of N~~Ras was induced in PC12 clones
stabley transfected with the N~~Ras gene by the addition of
dexamethasone to the starvation media. N~~Ras expression
inhibited the activation of MEKK by EGF as measured by its
ability to phosphorylate kinase inactive MEK. EGF-mediated
activation of Raf-B was also greatly reduced in N~~Ras
expressing PC12 cells compared to uninduced N~~Ras
transfectants. Addition of dexamethasone to wild type PC12
cells had no effect on the magnitude of MEKK or Raf-B
activation elicited by EGF. PC12 cell clones stably
transfected with the N~~Ras gene are less responsive to EGF-
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mediated activation of MEKK activity than are wild type
PC12 cells. These results indicate that functional Ras is
required for growth factor stimulated activation of both
Raf-B and MEKK in PC12 cells, suggesting that Ras may
mediate its effects on cell growth and differentiation
through the activation of multiple protein kinase effectors
from both the Raf and MEKK families. Thus, EGF stimulated
a peak of MEKK activity within 5 minutes which persisted
for at least 30 minutes following treatment, and was
similar to the timecourse of Raf-B activation. Nerve
growth factor (NGF) and the phorbol ester TPA also
activated MEKK, although to a lesser degree than EGF. MEKK
activity in immunoprecipitates or column fractions was
dissociable from that of EGF-stimulated c-Raf-1 and Raf-B
activities. Forskolin pretreatment abolished both MEKK and
Raf-B activation by EGF, NGF, and TPA (Fig. 18). Both MEKK
and Raf-B activation in response to EGF was inhibited by
stable expression of dominant negative N~~ Ras. These
findings represent the first demonstration of Ras-dependent
MEICK regulation by growth factors and suggest the emergence
of a complex intracellular kinase network in which Ras may
alternately couple between members of the Raf and MEKK
families.
To determine whether the growth factor-mediated
activation of 98 kD PC12 cell MEKK was inhibited by PKA,
forskolin was used to elevate intracellular cAMP and
activate PKA. Serum-starved PC12 cells were pretreated
with or without forskolin (50~tM) for 3 minutes to activate
i
2.1 ~~5~6
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protein kinase A and then with EGF (30ng/ml), NGF
(100ng/ml), or TPA (200nM) for 5 minutes and MEKK was
immunoprecipitated from equal amounts of soluble protein
from cell lysates and incubated with purified recombinant
catalytically inactive MEK-1 and (Y 3zp)ATP as described
above. Raf-B activity was also assayed from the same cell
lysates to test whether its regulation differed from that
of MEKK. Raf-B was immunoprecipitated from the same cell
lysates as described above and assayed for its ability to
phosphorylate MEK-1 as described above. Forskolin
pretreatment abolished the activation of both MEKK and Raf-
B by EGF, NGF, and TPA, as measured by their ability to
phosphorylate kinase-inactive MEK-1 (Fig. 18). Forskolin
treatment alone had no appreciable effect on either kinase.
These results demonstrate that in addition to Raf-1 and
Raf-B, PKA activation inhibits growth factor stimulation of
98 kD PC12 cell MEKK, suggesting the existence of a common
regulatory control point for PKA action which lies between
or downstream of Ras and upstream or at the level of each
of these three kinases.
Example 14
This Example describes the determination of whether a
similar or distinct MEK activity is involved in activation
of MAPK though G~ protein coupled receptors by measuring MEK
activity in cell lysates from thrombin stimulated Rat la
cells.
Thrombin stimulated cells exhibited a MEK activity
which co-fractionated with the major MEK peak detected in
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EGF stimulated cells. The magnitude of MEK activity from
thrombin challenged cells was generally two to three-fold
less than that observed with EGF stimulation, which
correlates with the smaller MAPK response the present
inventors have observed in thrombin challenged cells.
Differential regulation of MEK in Rat la and NIH3T3
cells expressing gip2, v-src, v-ras, or v-raf led the
present inventor to investigate the protein kinases that
are putative regulators of MEK-1. Recently, it was shown
that Raf-1 can phosphorylate and activate MEK. Raf
activation was assayed in the following manner. Cells were
serum starved and challenged in the presence or absence of
the appropriate growth factors, as described above. Serum
starved Rat la cells were c:-rallenged with buffer alone or
with EGF and Raf was immunoprecipitated using an antibody
recognizing the C terminus of Raf. Cells were lysed by
scraping in ice cold RIPA buffer (50 mM Tris, pH 7.2, 150
mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1.0% Triton X
100, 10 mM sodium pyrophosphate, 25 mM sodium
glycerophosphate, 2mM sodium vanadate, 2.1 fr.g/ml aprotinin)
and were microfuged for 10 min to remove nuclei. The
supernatants were normalized for protein content and
precleared with protein A Sepharose* prior to
immunoprecipitation with rabbit antiserum to the C terminus
of Raf-1 and protein A Sepharose for 2-3 h at 4°C. The
beads were washed twice with ice cold RIPA and twice with
PAN (10 mM Pipes, pH 7.0, lOOmM NaCl, 21).cg/ml aprotinin).
A portion of the immunoprecipitate was diluted with SDS
*trademark
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sample buffer and used for immunoblot analysis. The
remainder was resuspended in kinase buffer (20 mM Pipes pH
7.0, 10 mM MnClz, 150 ng kinase-inactive MEK-1, 30 ~Ci y-32P-
ATP and 20 Ecg/ml aprotinin) in a final volume of 50 M,1 for
30 min at 30°C. Wild type recombinant MEK-1 was
autophosphorylated in parallel as a marker. Reactions were
terminated by the addition of 12.5~u,1 5X SDS sample buffer,
boiled for 5 minutes and subjected to SDS-PAGE and
autoradiography.
The immunoprecipitated Raf, in the presence of Y-3zP-
ATP, was able to phosphorylate MEK-1. The recombinant MEK-
1 used in this assay was kinase inactive to ensure it did
not autophosphorylate as is observed with wild type MEK-1.
Little or no phosphorylation of MEK-1 by Raf was observed
in immunoprecipitates from control cells. EGF challenge
clearly stimulated Raf catalyzed phosphorylation of MEK-1;
in contrast, thrombin challenge of Rat la cells did not
measurably activate Raf even though endogenous MEK was
clearly activated. EGF stimulated Raf phosphorylation of
recombinant MEK-1 by approximately 2.6-fold over basal.
Little phosphorylation of MEK by Raf was observed in Raf
immunoprecipitates from Gip2 or v-Src expressing Rat la
cells. EGF stimulation was still capable of activating Raf
catalyzed phosphorylation of MEK-1 in these cell lines by
1.8 and 1.4-fold, respectively. The blunting of the EFG
response in Gip2 and v-Src expressing cells is likely a
result of desensitization of the EFG receptor upon
constitutive activation of MAPK. The amount of Raf in the
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immunoprecipitates was shown to be similar by subsequent
SDS-PAGE and immunoblotting using Raf antibody. Since
thrombin stimulation of MEK is two to three-fold over
basal, at least a 1.5-fold stimulation of MEK
phosphorylation is expected if Raf significantly
contributed to MEK activation by this growth factor. This
level of activation was detectable in the EGF stimulated
Gip2 and v-Src expressing cells lines. Thus, it is
unlikely that the failure to detect thrombin activation of
Raf is due to the sensitivity of the assay. Thrombin
stimulation of MAPK is maximal at 3 minutes. Stimulation
of Rat la cells for 1 or 5 minutes with thrombin did not
increase Raf activity.
In NIH3T3 cells, as in Rat la cells, EGF activates Raf
approximately 2.7-fold, while thrombin does not. V-Raf
expressing NIH3T3 cells showed no increase in MEK-1
phosphorylation.~ This result was unexpected since MEK was
clearly activated in v-Raf expressing NIH3T3 cells. Both
the p90 and p75 gag-raf fusion proteins in addition to c
Raf-1 were immunoprecipitated from v-Raf NIH3T3 cells by
the antisera. P75gag-raf has been shown to exhibit protein
kinase activity, but it is possible that the NHZ teraninal
gag fusion protein sterically hinders Raf phosphorylation
of recombinant MEK-1 in the in vitro assay system. Further
studies will have to be done to measure v-Raf kinase
activity. The results argue that activation of MEK cannot
be accounted for exclusively by the activation of Raf.
Additional regulatory kinases for MEK must exist which
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contribute to MEK activation in thrombin stimulated, G~
protein coupled pathways and in gip2 and v-src transfected
cells.
Example 15
This Example demonstrates the ability of a PPPSS-trunc
and Ncoi-trunc of MEKK protein to activate MAPK activity
compared with full-length MEKK protein and a negative
control protein.
The results shown in Fig. 19 indicate that the
truncated MEKK molecules were more active than the full-
length MEKK. Indeed, the truncated MEKK molecules were at
least about 1.5 times more active than full-length MEKK
protein. Thus, removal of the regulatory domain of MEKK
deregulates the activity of the catalytic domain resulting
in improved enzyme activity.
Example 16
This example describes the preferential activation of
JNK by MEKK compared with Raf.
HeLa cells were transiently transfected with truncated
MEKK3~o_~$ under control of an inducible mammary tumor virus
promoter, together with epitope tagged JNK1 (described in
detail in Derijard et al., p. 1028, 1994, Cell, Vol. 76).
Other HeLa cells were also transiently transfected with
truncated BXB-Raf under control of an inducible mammary
tumor virus promoter, together with epitope tagged JNK1
(Derijard et al., ibid.) . The following day, MEKK3~o_~8
expression and BXB-Raf expression were induced by
administration of dexamethasone (10 ~,M) for 17 hours. Cell
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extracts were then prepared and assayed for JNK activity
using an immune complex kinase assay (detailed in Derijard
et al., ibid.). Phosphorylation was quantitated by
phosphorimager analysis. The results shown in Fig. 20
indicate that MEKK stimulated about 30-fold to about 50-
fold activation more JNK activity over unstimulated cells
(basal) and about 15-fold to about 25-fold JNK activity
over Raf stimulated cells.
Example 17
This example describes that the phosphorylation of c-
Myc transactivation domain in response to MEKK expression
activates MYC-GAL 4 transcriptional activity.
Two separate expression plasmids were constructed as
follows. The expression plasmid pLNCX was ligated to a
cDNA clone comprising c-myc (1-103) ligated to GAL4 (1-147)
(Seth et al., pp. 23521-23524, 1993, J. Biol. Chem., Vol.
266) to form the recombinant molecule pMYC-GAL 4. The
expression plasmid UAS~-TK Luciferase (Sadowski et al., pp.
563-564, 1988, Nature, Vol. 335) was transfected with
either pMYC-GAL 4 or pLU-GAL into Swiss 3T3 cells using
standard methods in the art to form recombinant cells
herein referred to as LU/GAL cells. Recombinant control
cells were also produced by transfecting in pGAL4-Control
plasmids containing GAL4 (1-147) alone in the absence of c
myc (1-103).
LU/Gal cells were transfected with either pMEKK37o-r38~
pMEKK (encoding full-length MEKK~_~8), BXB-Raf, pMyc-Gal4,
pCREB-Gal4 (encoding CREB~_Z6~ fused to Gal 4~_~4~; Hoeffler et
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al., pp. 868-880, 1989, Mol. Endocrinol., Vol. 3), pGal4,
or CREB fusion protein referred to as GAL4.
The transfected cells were incubated overnight and
then lysed using methods standard in the art. The
luciferase activity of each cell lysate was measure on a
luminometer. The results shown in Fig. 21 indicate that
MEKK is selectively capable of stimulating the
phQsphorylation of c-Myc transactivation domain in such a
manner that the c-Myc domain is activated and induces
transcription of the transfected luciferase gene. In
addition, the results indicate that MEKK does not stimulate
CREB activation. Also, activated Raf is unable to
stimulate Myc activation. A schematic representation of
the activation mechanism of c-Myc protein by MEKK is shown
in Fig. 23.
Example 18
This Example describes the phosphorylation of p38 MAPK
protein by MEKK 3 protein and not MEKK 1 protein.
COS cells were transfected with the expression plasmid
pCVM5 ligated to cDNA clones encoding either MEKK 1 or MEKK
3 protein, or a control pCVMS plasmid lacking MEKK cDNA
inserts. Forty-eight hours after transfection, the COS
cells were lysed and the lysate fractionated on a Mono Q
FPLC column using conditions described in Example 4. The
fractions were analyzed for tyrosine phosphorylation of MAP
kinase-like enzymes using the kinase assay described in
Example 4. Referring to Fig. 23, expression of MEKK 3
induces tyrosine phosphorylation of p38 MAPK and the p42
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and p44 forms of MAPK. MEKK 1, however, only induces weak
phosphorylation of p38 MAPK but does induce phosphorylation
of p42 and p44 MAPK.
Example 19
This example describes MEKK-induced apoptosis.
Cells were prepared for the apoptosis studies as
follows. Swiss 3T3 cells and REF52 cells were transfected
with an expression plasmid encoding B-Galactoctosidase (B-
Gal) detection of injected cells. One set of B-Gal
transfected cells were then microinjected with an
expression vector encoding MEKK3~o_~$ protein. Another set
of B-Gal transfected cells were then microinjected with an
expression vector encoding truncated BXB-Raf protein.
A. Beauvericin-induced anoptosis
A first group of transfected Swiss 3T3 cells and REF52
cells were treated with 50 ~,M beauvericin for 6 hours at
37°C. Beauvericin is a compound known to induce apoptosis
in mammalian cells. A second group of cells were treated
with a control buffer lacking beauvericin. The treated
cells were then fixed in paraformaldehyde and permeabilized
with saponin using protocols standard in the art. The
permeabilized cells were then labelled by incubating the
cells with a fluorescein-labelled anti-tubulin antibody
(1:500; obtained from GIBCO, Gaithersburg, MD) to detect
cytoplasmic shrinkage or 10 ~M propidium iodide (obtained
from Sigma, St. Louis, MO) to stain DNA to detect nuclear
condensation. The labelled cells were then viewed by
differential fluorescent imaging using a Nikon Diaphot
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fluorescent microscope. Fig. 24 shows two fields of Swiss
3T3 cells and REF52 cells, one field representing cells
treated with the control buffer and a second field
representing cells treated with beauvericin. The cells
treated with beauvericin demonstrated cytoplasmic shrinkage
(monitored by the anti-tubulin antibodies) and nuclear
condensation (monitored by the propidium iodide)
characteristic of apoptosis.
B. MEKK-induced apoptosis
Swiss 3T3 cells and REF52 cells microinjected with a
B-galatoctosidase expression plasmid, and an MEKK encoding
plasmid or a BXB-Raf encoding plasmid, were treated and
viewed using the method described above in Section A. An
anti-B-Gal antibody (1:500, obtained from GIBCO,
Gaithersburg MD) was used to detect injected cells.
Referring to Fig. 25, microscopic analysis of REF52 cells
indicated that the cells expressing MEKK protein underwent
cytoplasmic shrinkage and nuclear condensation leading to
apoptotic death. In contrast, cells expressing BXB-Raf
protein displayed normal morphology and did not undergo
apoptosis. Similarly, referring to Fig. 26, microscopic
analysis of Swiss 3T3 cells indicated that the cells
expressing MEKK protein underwent cytoplasmic shrinkage and
nuclear condensation leading to apoptotic death. In
contrast, cells expressing BXB-Raf protein displayed normal
morphology and did not undergo apoptosis.
Fig. 27 shows 3 representative fields of RFE52 cells
expressing MEKK protein which have undergone substantial
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cytoplasmic shrinkage and nuclear condensation compared
with a control cell not expressing MEKK. Similarly, Fig.
28 shows 3 representative fields of Swiss 3T3 cells
expressing MEKK protein which have undergone substantial
cytoplasmic shrinkage and nuclear condensation compared
with a control cell not expressing MEKK. Thus, MEKK and
not Raf protein can induce apoptotic programmed cell death.
Example 20
This Example describes regulation of MAPK activity by
both MEKK and Raf protein.
COS cells were prepared using the method described in
Example 3. In addition, COS cells were transfected with
the pCVMVS Raf construct (1 ~Cg: Raf). FPLC MONO Q ion-
exchange column fractions were prepared as described in
Example 3 and assayed for MAPK activity according to the
method described in Heasley et al., ibid.
Referring to Fig. 29, both MEKK and Raf overexpression
in COS 1 cells resulted in similar levels of stimulation of
MAPK activity over basal levels.
The foregoing description of the invention has been
presented for purposes of illustration and description.
Further, the description is not intended to limit the
invention to the form disclosed herein. Consequently,
variations and modifications commensurate with the above
teachings, and the skill or knowledge in the relevant art
are within the scope of the present invention. The
preferred embodiment described herein above is further
intended to explain the best mode known of practicing the
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invention and to enable others skilled in the art to
utilize the invention in various embodiments and with
various modifications required by their particular
applications or uses of the invention. It is intended that
the appended claims be construed to include alternate
embodiments to the extent permitted by the prior art.
