Note: Descriptions are shown in the official language in which they were submitted.
W095/21923 2 1 8 2 9 6 7 PCTI~b5S~272
P~vu~llON AND ~SE OF MAP ~T~ P~OS
AND ENCODING N~CLEIC ACID lH~ On
The present invention relates to phosphatases. In
particular, it relates to polypeptides having MAP kinase
phosphatase activity, encoding nucleic acid therefor,
antibodies thereto, and methods of production and use of
the phosphatases, encoding nucleic acid and antibodies.
It also relates to screens for substances which have an
effect on phosphatase activity and screens for MAP kinase
phosphatase polypeptides and genes. Additionally, it
relates to methods of diagnosis and treatment for
proliferative diseases involving loss of MAP kinase
phosphatase function.
The mechanism by which extracellular signals for
growth and differentiation are transmitted to the nucleus
to alter gene expression is the subject of much current
investigation. In many cases, the transduction of these
signals requires the activities of key enzymes known
generally as "Mitogen activated protein (MAP) kinases~.
MAP kinase pathways have been implicated in a large
number of signal transduction pathways. For instance,
activation of MAP kinases has been observed during growth
factor stimulation of DNA synthesis and during
differentiation, secretion and stimulation of glycogen
synthesis (1). MAP kinase has been shown to
phosphorylate and activate effector substrates such as
the transcription factors c-jun and elk-l. For a summary
2 1 82967
WOg5/21923 PCT/GB95/00272
of MAP kinases and pathways in which they are known to be
involved, see a review by Roger Davis (50).
MAP kinase is activated by phosphorylation on
threonine and tyrosine by a dual specificity kinase, "MAP
kinase kinase". This kinase kinase is in turn activated
by phosphorylation by "MAP kinase kinase kinase", one
form of which is the proto-oncogene c-raf. The
activation of c-raf is not fully understood at present
but apparently there is a requirement for an interaction
with GTP-bound p21 ras protein (2).
The full picture of how MAP kinase pathways are
switched off is as yet unclear. Down-regulation of MAP
kinase activity by de-phosphorylation is likely to be of
key importance. The human gene CL100 (3) and its murine
homologue 3CH134 (Charles et al, 1992) were originally
discovered as genes whose transcription was stimulated by
growth factors, oxidative stress and heat shock.
Subsequently, they were shown to encode polypeptides that
have both serine/threonine and tyrosine phosphatase
activity (5 & 6). This removal of phosphate from both
threonine and tyrosine on MAP kinase is unusual. When
expressed in vitro (6) this gene product has been shown
to be very specific for MAP kinase and leads to its
inactivation. Co-expression of the murine gene 3CH134
and the erk2 MAP kinase isoform in m~mm~l ian cells leads
to the dephosphorylation and inactivation of the MAP
kinase (7). Furthermore, it has been shown recently that
this phosphatase gene can also block cellular DNA
`` 21 82q67
WO95/21923 PCT/GB95/00272
synthesis induced by an activated version of the ras
oncogene in rat embryo fibroblasts (51).
The present invention has resulted from the
surprising discovery of several new genes, each encoding
a polypeptide implicated in MAP kinase regulatory
systems.
For present purposes, the terms "Mitogen-activated
protein kinase", "MAP kinase" and "MAPK" apply to protein
kinases that are activated by dual phosphorylation on
threonine and tyrosine. This may be in reponse to a wide
array of stimuli. Different MAP kinases are activated in
repsonse to different extracellular stimuli, including
(depending on the MAPK) stress, osmotic stress, mating
pheromone (in yeast), growth factors, TNF, IL-l and LPS.
MAP kinases include SMKl, HOGl, MPKl, FUS3/KSSl, spkl,
ERKl/ERK2, JNK/SAPK, p38. "MAP kinase phosphatase"
activity or function is the ability to dephosphorylate
one or preferably both of the threonine and tyrosine
residues on a MAP kinase, which residues are
phosphorylated in the activation of the MAP kinase. Put
another way, MAP kinase phosphatases are capable of
hydrolysing either or preferably both phosphothreonine
and phosphotyrosine residues on a MAP kinase.
Signalling by protein tyrosine phosphatases (PTPs).
The mechanism by which extracellular signals for
growth and differentiation are transmitted to the nucleus
to alter gene expression is currently the subject of much
WOgS/21923 2 1 8 2 9 6 7 PCT/GB9S/00272
investigation. Tyrosine phosphorylation plays a central
role in these events [8,9], and is regulated by opposing
activities of kinases and phosphatases. Although
phosphatases may act directly by dephosphorylation of
protein tyrosine kinase (PTK) receptors, it can be
envisaged that they dephosphorylate the sig,nalling
molecules downstream, since PTK receptors are regulated
at least in part through internalisation.
Increasing attention has been focused on the
expanding family of protein tyrosine phosphatases which
can be categorised as receptor-like and nonreceptor
molecules [10 - 12]. Deregulated expression of some
nontransmembrane tyrosine phosphatases has been shown to
affect cell growth and increase the proportion of
multinucleated cells [13,14]. Evidence suggests that
PTPs may function as negative regulators of cell
proliferation [15,18].
This is supported by the observations that PTPase
inhibitors are able transiently to substitute for growth
factors and induce mitogenic response [19,20].
Furthermore, it has been ~mo~qtrated that overexpression
of PTPs can revert the transformed phenotype of v-src and
suppress subsequent transformation by both the oncogenes
neu and v-erbB [21,22]. However, it is apparent that
PTPs are able to promote growth stimulatory effects [23],
so a critical balance must exist in the cell between
these activities to ensure proper growth control.
W095/21923 ~ ; 2 1 8 2 9 6 7 pcTlGs9~loo272
Signal transduction pathways regulating M~P kinases
A key element in signal transduction from an
activated receptor tyrosine kinase to an intracellular
response is now recognised to involve the family of MAP-
kinases, pathways implicated in many diverse cell types
[1]. Two forms of MAP kinase have been purified from
human fibroblasts with molecular weights p42~Pk and
p44~pk, (ERK-2 and ERK-1 respectively), [24]. Activation
requires an ordered phosphorylation of a threonine and
tyrosine located within the conserved kinase subdomain 8,
(T183, Y185), [25,26].
The use of dominant-negative mutations and homology
experiments in yeast have proved to be invaluable tools
in the elucidation of this signal cascade. Evidence
suggests that in response to growth factor activated
PTKs, a pre-existing Grb2-SOS 1 complex binds to tyrosyl
phosphorylated Shc through SH2 domains, (27), thus
recruiting Ras activator molecules to the plasma membrane
[28,29]. Alternatively, Grb2 may directly interact with
autophosphorylated receptors. Extensive studies support
hypotheses that signals converge through Ras [30,32], and
continue through the Ser/Thr kinase Raf [33,35]. The
activation of Raf-1 is not fully understood at present
but apparently there is a requirement for an interaction
with GTP-bound p21 ras protein [36,37]. The use of
oncogenic forms of Raf-1 have shown it to act as a
putative MKKK [33-35], along with MEKK [38], and c-Mos
[39,40], which results in sequential activation of
WO95/21923 2 1 g 2 q 6 7 PCT/GB95/00272
Scr/Thr kinases MEK [24,41], which ultimately
phosphorylate the MAP kinases, see Figure 1.
Recently several new members of the MAP kinase gene
family have been discovered (50). These kinases, called
JNK and p38 are involved in a variety of cellular
responses. JNK is activated by stress, Tumour Necrosis
Factor (TNF), Interleukin-1 (IL-1) and ultra-violet (W)
light. p38 is activated by lipopolysaccharide (LPS).
Both kinases are related to the erk-type MAP kinases in
that they-are activated by phosphorylation on both
tyrosine and threonine. Deactivation by phosphatases is
indicated.
A novel subfamily of Protein Phosphatases
Pathways have been defined involving cascades of
protein phosphorylation capable of inducing a complex set
of immediate early genes, functionally significant in
cell cycle regulation and oncogenic transformation. An
essential feature of these phosphorylation events is
their reversibility, and indeed tyrosine phosphorylation
is often transient. It is known that removal of
phosphate from either threonine by PP2A or from tyrosine
by CD45 results in loss of MAP kinase activity [25]
though how exactly this pathway is switched off in vivo
is yet to be identified.
The present invention is founded on the discovery
and isolation of several nucleic acid molecules encoding
proteins which are related to the known MAP kinase
WOgS/21923 ~ 2 1 8 2 9 6 7 pcTlGBs~loo272
phosphatases. Using insight gained from specialist
knowledge in the field, the inventors were able to design
an investigative procedure which resulted in the
obtention of the new genes. The actual procedure used is
described in detail below.
The sequences of the polypeptides encoded by the
novel nucleic acid sequences share a degree of homology
with the sequence of the known MAP kinase phosphatase,
CLlO0, which is sufficient for indication as
phosphatases, particularly MAP kinase phosphatases. This
is interesting and useful:
MAP kinase phosphatases are likely to act as off
switches for cell proliferation. The fact that there are
multiple MAP kinase phosphatases suggests that there may
be some specificity to the off switches. Activators of
the MAP kinase phosphatases either general or for
specific family members may be anti-proliferative agents.
Provision of nucleic acid encoding phosphatases enables
screening for such activators. Loss of MAP kinase
phosphatase activity by, for example, mutation may lead
to uncontrolled cell proliferation. Hence, some of these
genes may prove to be "tumour suppressor genes".
In addition to MAP kinase, the phosphatases may have
novel substrates. These substrates may also be key
regulators of cell proliferation and potential targets
for intervention by drug inhibitors.
The provision of various MAP kinase phosphatases and
encoding nucleic acid therefor enables the production of
WOgS/21923 2 1 8 2 9 6 7 PCT/GB95/00272
antibodies able to bind, or specific for, the phosphatase
polypeptides. Such antibodies are useful in the
determination of the presence of a phosphatase in a test
sample, e.g. containing tissue or cellular material, for
example to determine some abnormality in the level or
nature of the polypeptide. Antibodies able to
discriminate between normal and abnormal molecules may be
used in a diagnostic or screening context, e.g. in the
determination of the underlying cause of a proliferative
disorder such as a tumour.
Similarly, nucleic acid probes may be used in
screening nucleic acid from cells of an individual, for
example to determine whether those cells contain the
wild-type gene encoding a particular phosphatase, and if
they do whether they are homozygous or heterozygous.
Since MAP kinase phosphatases are involved in
deactivation of MAP kinases, they are likely to have
tumour suppressor function such that the absence of wild-
type may have adverse effects on control of cell
proliferation and heterozygosity may predispose an
indi~idual to a proliferative disorder. Thus important
clinical information may be obtained, enabling
appropriate therapeutic action to be taken.
Nucleic acid encoding a MAP kinase phosphatase may
be used in a therapeutic context to counter the effect of
loss of normal MAP kinase phosphatase activity in cells.
Loss of such activity, which may be total or partial, may
lead to a proliferative disorder wherein normal
WO95/21923 ~ ~ 2 1 8 2 9 6 7 pcTlGs95loo272
regulation of cell growth is disrupted. Uncontrolled
cell growth, i.e. cell growth which is not properly
controlled, is involved in numerous disorders, both
malignant (cancer) and benign. Gene therapy using one or
more MAP kinase phosphatase-encoding nucleic acid
molecules may be used in amelioration of disorders
resulting from a loss of normal MAP kinase phosphatase
activity.
Sequence information is presented in the
accompanying figures, discussed below, along with
experimental protocols and results ~emonqtrating MAP
kinase phosphatase ac~ivity. As of 9 February 1995, none
of the sequences are present in the EMBL/GENBANK
database.
According to one aspect of the present invention
there is provided a nucleic acid molecule comprising a
sequence of nucleotides encoding a polypeptide which
comprises a sequence of amino acids encoded by nucleic
acid with any one of the encoding sequences shown in
Figure 2. The nucleic acid molecule may comprise any of
the sequences shown in Figure 2 or may comprise a
sequence which is a mutant, derivative or allele of the
sequences shown. The sequence may differ from any of
those shown by a change which is addition, insertion,
deletion or substitution of one or more nucleotides of
any of the sequences shown. Changes to a nucleotide
sequence may result in an amino acid change at the
protein level, or not, as determined by the genetic code.
woss/2l923 2 1 8 2 q 6 7 PCT/GB95/00272
Thus, nucleic acid according to the present invention may
comprise a sequence different from any of the sequences
shown in Figure 2, yet encode a polypeptide with the same
amino acid sequence as any of those shown sequences. On
the other hand the encoded polypeptide may comprise an
amino acid sequence which differs by one or more amino
acid residues from any of those encoded by the encoding
sequences shown in Figure 2.
Also provided by the present invention are a vector
comprising nucleic acid as set out above, particularly
any expression vector from which the encoded polypeptide
can be expressed under appropriate conditions, and a host
cell cont~;n;ng any such vector or nucleic acid. An
expression vector in this context is a nucleic acid
molecule comprising nucleic acid encoding a polypeptide
of interest and appropriate regulatory sequences for
expression of the polypeptide, either in an in vi tro
expression system, e.g. reticulocyte lysate, or in vivo,
e.g. in eukaryotic cells such as COS or CHO cells or in
prokaryotic cells such as E. coli .
Nucleic acid according to the present invention may
be isolated (an "isolate") in the sense of being removed
from its natural environment, or free from other nucleic
acid obtainable from the same species (e.g. encoding
another polypeptide). Of course, nucleic acid according
to the present invention may be wholly or partially
synthetic.
The present invention also provides a polypeptide
WO9S/21923 2 1 8 2 9 6 7 PCT/~b5S/~272
11
having MAP kinase phosphatase activity and comprising a
sequence of amino acids encoded by nucleic acid with any
one of the encoding sequences shown in Fiyure 2. Amino
acid sequences encoded by the sequences of Figure 2
S appear in Figure 3.
Variants, mutants or derivatives of these
polypeptides, especially but not necessarily those which
retain MAP kinase phosphatase activity, (eg variants
resulting from insertion, deletion or substitution of one
or more amino acids) are also encompassed by the present
invention. Variant, mutant or derivative polypeptides
lacking MAP kinase phosphatase activity may be useful,
particularly if they retain ability to interact or bind
with MAP kinase. For example, tyrosine and dual
specificity phosphatases have a cysteine residue located
at the active site. Alteration of this cysteine to a
serine in CL100 and its murine homolog 3CH134 leads to
the abolition of catalytic activity (8). However,
expression of this catalytically dead form leads to an
increase in the phosphorylation of MAP kinase (ERK2).
The mutant/derivative forms a specific complex with ERK2
MAP kinase. Presumably this association blocks
dephosphorylation of ERK2 by endogenous CL100/3CH134.
Thus, also provided by the present invention is a
polypeptide comprising an amino acid sequence which
comprises an allele, derivative or mutant, by way of
addition, insertion, deletion or substitution of one or
more amino acids, of an amino acid sequence encoded by
WO95/21923 - 2 1 8 2 q 6 7 PCTIGB95/00272
nucleic acid with any one of the encoding sequences shown
in Figure 2.
A derivative is a substance derivable from a
polypeptide. The derivative may differ from a
polypeptide from which it may be derived by the addition,
deletion, substitution or insertion of one or more amino
acids, or the linkage or fusion of other molecules to the
polypeptide. Changes such as addition, deletion,
substitution or insertion may be made at the nucleotide
or protein level.
The provision of amino acid and nucleic acid
sequence information for various polypeptides with MAP
kinase phosphatase activity, the first ~emon.qtration that
a family of such polypeptides exists, enables the
obtention of other polypeptides and encoding nucleic acid
therefor having a significant degree of homology to the -
sequences given herein. Such homology might be greater
than about 70%, preferably greater than about 80~, more
preferably greater than about 85~ or about 90~ and most
preferably greater than about 95~. Homology between
orthologues, that the equivalent sequence in different
species, is very high, while homology between sequences
of a given species varies somewhat, as illustrated
herein.
According to a further aspect of the present
invention there is provided a polypeptide which has an
amino acid sequence which is homologous to a sequence of
amino acids encoded by nucleic acid with any one of the
:
WO95/21923 2 1 8 2 ~ 6 7 PCT/GD55/U~272
encoding sequences shown in Figure 2, and which has MAP
kinase phosphatase activity but is not CLlO0, or an
orthologue thereof. As discussed, the present invention
provides the first demonstration that a multitute of MAP
kinase phosphates exist. Previously, only CLlO0 and its
mouse orthologue 3CHl34 were known as MAP kinase
phosphatases obtainable from m~mm~l S. Prior to the
making of the present invention PAC-l (42) had been
identified as a T-cell specific protein. Since then,
this has been found to have MAP kinase phosphatase
activity. Accordingly, PAC-l, nucleic acid encoding it,
and so on may also be excluded from the present
invention. In one embodiment of the present invention
the homologous polypeptide is an orthologue of a
polypeptide comprising an amino acid sequence encoded by
a nucleotide sequence shown in Figure 2.
Of course, the present invention extends to variant
polypeptides of those naturally occuring polypeptides
homologous to those encoded by sequences shown in Figure
2, for example alleles, derivatives or mutants, wherein
there is addition, insertion, deletion or substitution of
one or more amino acids. These may or may not have MAP
kinase phosphatase activity, but preferably are at least
able to interact with or bind to a MAP kinase.
A convenient way of producing a polypeptide
according to the present invention is to express nucleic
acid encoding it.
Accordingly, the present invention also encompasses
2 1 82967
WO95/21923 PCT/~b95/~272
a method of making a polypeptide which has MAP kinase
phosphatase activity, the method comprising expression
from a vector which comprises nucleic acid encoding the
polypeptide, the nucleic acid comprising nucleic acid
encoding a polypeptide comprising an amino acid sequence
encoded by an encoding nucleotide sequence shown in
Figure 2. This may conveniently be achieved by growing a
host cell, cont~;n;ng such a vector, under conditions
which cause or allow expression of the polypeptide.
Polypeptides may also be expressed in in vitro systems,
such as reticulocyte lysate. Alleles, derivatives,
variants and mutants may be expressed likewise.
Systems for cloning and expression of a polypeptide
in a variety of different host cells are well known.
Suitable host cells include bacteria, eukaryotic cells
such as m~mm~l ian cells and yeast, and baculovirus
systems. ~mm~l ian cell lines available in the art for
expression of a heterologous polypeptide include Chinese
hamster ovary cells, HeLa cells, baby hamster kidney
cells, COS cells and many others. A common, preferred
bacterial host is E. coli.
Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including
promoter sequences, terminator fragments, polyadenylation
sequences, enhancer sequences, marker genes and other
sequences as appropriate. For further details see, for
example, Molecular Cloning: a Laboratory M~n~ 7: 2nd
edition, Sambrook et al, 1989, Cold Spring Harbor
WOgS/21923 2 1 8 2 9 6 7 PCT/~b551~^272
Laboratory Press. Transformation procedures depend on
the host used, but are well known.
The proteins provided by the present invention may
be purified from natural sources, or being produced
recombinantly. Such purified proteins and methods of
their purification, from natural sources or recombinantly
produced, are encompassed by the present invention.
The provision of novel polypeptides enables for the
first time the production of antibodies able to bind
them. Accordingly, a further aspect of the present
invention provides an antibody able to bind a polypeptide
disclosed herein. Such an antibody may be specific for
one or more of the polypeptides, in the sense of being
able to distinguish between a polypeptide it is able to
bind and other MAP kinase phosphatases which it is either
not able to bind or which it binds more weakly. Other
antibodies according to the present invention are able to
bind MAP kinase phosphatases generally.
Preferred antibodies according to the invention are
isolated, in the sense of being free from cont~min~nts
such as antibodies able to bind other polypeptides and/or
free of serum components. Monoclonal antibodies are
preferred for some purposes, though polyclonal antibodies
are within the scope of the present invention.
Antibodies may be obtained using techniques which
are standard in the art. Methods of producing antibodies
include immunising a m~mm~l (eg mouse, rat, rabbit,
horse, goat, sheep or monkey) with the protein or a
WO9S/21923 2 ~ 8 2 9 6 7 PCT/GB95/00272
16
fragment thereof. Antibodies may be obtained from
immunised animals using any of a variety of techniques
known in the art, and screened, preferably using binding
of antibody to antigen of interest. For instance,
Western blotting techniques or immunoprecipitation may be
used (Armitage et al, 1992, Nature 357: 80-82).
As an alternative or supplement to ;mml~n;sing a
m~mm~l with a peptide, an antibody specific for a protein
may be obtained from a recombinantly produced library of
expressed immunoglobulin variable domains, eg using
lambda bacteriophage or filamentous bacteriophage which
display functional immunoglobulin binding domains on
their surfaces; for instance see WO92/01047. The library
may be naive, that is constructed from sequences obtained
from an organism which has not been ;mml1n;sed with any of
the proteins (or fragments), or may be one constructed
using sequences obtained from an organism which has been
exposed to the antigen of interest.
Antibodies according to the present invention may be
modified in a number of ways. Indeed the term "antibody"
should be construed as covering any binding substance
having a binding domain with the required specificity.
Thus the invention covers antibody fragments,
derivatives, functional equivalents and homologues of
antibodies, including synthetic molecules and molecules
whose shape mimicks that of an antibody enabling it to
bind an antigen or epitope.
Example antibody fragments, capable of binding an
WO95/21923 ~ 2 t 8 2 q 6 7 PcT/Gsg5/00272
antigen or other binding partner are the Fab fragment
consisting of the VL, VH, Cl and CHl domains; the Fd
fragment consisting of the VH and CH1 domains; the Fv
fragment consisting of the VL and VH ~o~;n~ of a single
arm of an antibody; the dAb fragment which consists of a
VH domain; isolated CDR regions and F(ab')2 fragments, a
bivalent fragment comprising two Fab fragments linked by
a disulphide bridge at the hinge region. Single chain Fv
fragments are also included.
A hybridoma producing a monoclonal antibody
according to the present invention may be subject to
genetic mutation or other changes. It will further be
understood by those skilled in the art that a monoclonal
antibody can be subjected to the techniques of
recombinant DNA technology to produce other antibodies or
ch,~ric molecules which retain the specificity of the
original antibody. Such techniques may involve
introducing DNA encoding the immunoglobulin variable
region, or the complementarity determining regions-
( CDRs ), of an antibody to the constant regions, or
constant regions plus framework regions, of a different
immunoglobulin. See, for instance, EP184187At GB
2188638A or EP-A-0239400. Cloning and expression of
chimeric antibodies are described in EP-A-0120694 and EP-
A-0125023.
Hybridomas capable of producing antibody with
desired binding characteristics are within the scope of
the present invention, as are host cells, eukaryotic or
WO95/21923 2 1 8 2 9 6 7 pcTlGs9sloo272
prokaryotic, containing nucleic acid encoding antibodies
(including antibody fragments) and capable of their
expression. The invention also provides methods of
production of the antibodies comprising growing a cell
capable of producing the antibody under conditions in
which the antibody is produced, and preferably secreted.
Antibodies according to the present invention may be
used in screening for the presence of a polypeptide, for
example in a test sample comprising cells or cell lysate.
Similar proposals have been made for the known tumour
suppressor gene retinoblastoma ~e.g. in WO94/01467 and
AU-A-52461/90).
For instance, a particular antibody may be able to
distinguish between a wild-type polypeptide and a
corresponding polypeptide with some difference in one or
more epitopes as a result in a variation in amino acid
sequence. The presence of a particular epitope may be
indicative of a loss of MAP kinase phosphatase activity,
or otherwise predictive of susceptibility or
predisposition to a proliferative disorder. Similarly,
antibodies may be used to determine the presence of wild-
type polypeptide in cases wherein loss of expression of
the polypeptide is predictive of poor patient prognosis
or susceptibility to a proliferative disorder.
Antibodies may be used to determine whether cells of
a tumour lack or have aberrerant MAP kinase phosphatase
activity, e.g. because of mutation of the polypeptide or
loss of expression of the polypeptide.
Wo95121923 ~ ~: 2 1 8 ~ 9 b 7 PCT/GBg5/00272
19
Antibody binding to a sample may conveniently be
determined by employing a suitable labelling system for
the antibody. Numerous approaches and labels are well
known in the art, especially for immunoassays. Labelling
may be direct or indirect. Detectable labels may be any
substance having a physical or chemical property which
may be detected, including enzymatic groups such as
alkaline phosphatase and peroxidases, fluorescers,
chromophores, luminescers and radioisotopes. Biotin and
avidin/streptavidin systems may be employed.
Particularly suitable is the avidin-biotin complex-
immunoperoxidase technique described by Cordon-Cardo et
al (Amer. J. Pathol. 126: 269-284, 1987).
Antibodies according to the present invention may
additionally be used in isolating or purifying any
polypeptide as disclosed herein, including mutants,
alleles, derivatives etc, according to standard
techniques.
The new genes were isolated using a combination of
PCR and low stringency hybridisation analysis. The
primers used in the PCR had the sequences
TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A)CC(A,T)GT(A,G,T)
GA and
AT(G,C,T)CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC, and
were designed based on amino acid sequences, YDQGGPVE and
VHCQAGI conserved between human and mouse CL100 and the
human PAC-1 gene (42). (At the time of making the
present invention, PAC-1 was not known to have MAP kinase
WO95/21923 2 1 8 2 9 6 7 PCT/GBgS/00272
phosphatase activity.)
A further aspect of the present invention provides
an oligonucleotide with one of these sequences, a
sequence complementary to one of these, for use in a
method of obtaining nucleic acid encoding a protein with
phosphatase activity, particularly MAP kinase phosphatase
activity, comprising hybridisation of two primers to
target nucleic acid. The hybridisation may be as part of
a PCR procedure, or as part of a probing procedure not
involving PCR. An example procedure would be a
combination of PCR and low stringency hybridisation
comparable to that used by the present inventors. A
screening procedure, chosen from the many available to
those skilled in the art, is used to identify successful
hybridisation events and isolated hybridised nucleic
acid.
The sequences provided in Figure 2 are themselves
useful for identifying nucleic acid encoding other
phosphatase proteins, such as those with MAP kinase
phosphatase activity. Accordingly, the present invention
provides a method of obt~;n;ng nucleic acid encoding a
protein with phosphatase activity, particularly a protein
with MAP kinase phosphatase activity, the method
comprising hybridisation of a probe having any of the
sequences shown in Figure 2 or a complementary sequence,
to target nucleic acid. Hybridisation is generally
followed by identification of successful hybridisation
and isolation of nucleic acid which has hybridised to the
WO95/21923 2 1 8 2 9 6 7 PCT/GB95/00272
21
probe. The method may involve one or more steps of PCR.
It will not always be necessary to use a probe with
one of the complete sequences shown in the figures.
Shorter fragments, particularly fragments with a sequence
conserved between two or more of the sequences, may be
used. Nucleic acid which has some alteration, eg
insertion, deletion or substitution of one or more
nucleotides, in the sequence will be useful, provided the
degree of homology with one of the sequence given is
O sufficiently high.
A nucleic acid probe with a sequence selected from:
(i) TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A)CC(A,T)
GT(A,G,T)GA;
(ii) AT(G,C,T)CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC;
(iii) a sequence complementary to (i) or (ii);
(iv) any one of the nucleotide sequences shown in Figure
2;
(v) a nucleotide sequence complementary to any one of
the sequences shown in Figure 2;
0 (vi) a nucleotide sequence which comprises an allele,
derivative or mutant, by way of addition, insertion,
deletion or substitution of one or more nucleotides,
of any one of the sequences shown in Figure 2, or a
nucleotide sequence complementary thereto; and
5 (vii) a nucleotide sequence which is a fragment of
any one of (v), (vi) and (vii);
may equally be used in a method of screening cells for
the presence of nucleic acid encoding a polypeptide
WOg5/21923 ` PCT/GB95/00272
21 82967
comprising a sequence of amino acids encoded by nucleic
acid with any one of the encoding sequences shown in
Figure 2, or an allele, derivative or mutant, by way of
addition, insertion, deletion or substitution of one or
more nucleotides, of any one of the encoding sequences
shown in Figure 2, the method comprising hybridising such
a nucleic acid probe to a sample of nucleic acid of the
cells and determining binding of the probe to the sample.
Where the nucleic acid is double-stranded DNA,
hybridisation will generally be preceded by denaturing to
produce single-stranded DNA. Probing may employ the
standard Southern blotting technique. For instance DNA
may be extracted from cells of interest (e.g. from
normal, suspect or tumour tissue) and digested with
different restriction enzymes. Restriction fragments may
then be separated by electrophoresis on an agarose gel,
before denaturationg and transfer to a nitrocellulose
filter. Labelled probe may be hybridised to the DNA
fragments on the filter and binding determined.
Binding of a probe to target nucleic acid (e.g. DNA)
may be measured using any of a variety of techniques at
the disposal of those skilled in the art. For instance,
probes may be radioactively, fluorescently or
enzymatically labelled. Other methods not employing
labelling of probe include eX~mtn~tion of restriction
fragment length polymorphisms, amplification using PCR,
RNAase cleavage and allele specific oligonucleotide
probing.
WO9S/21923 2 1 8 2 9 ~ 7 pcTlGs9sloo272
23
Abnormalities in binding of the probe to target DNA
from an individual's cells may indicate that the
individual is susceptible to a cell proliferation
disorder arising from aberrant MAP kinase phosphatase
function. Thus, individuals may be screened for the
presence of one or more copies of a MAP kinase
phosphatase gene to assist in assessing likelihood of
developing a disorder involving uncontrolled cell
proliferation. The screening may be prenatal.
Similarly, tumours may be identified as having resulted
from a loss of MAP kinase phosphatase function if probe
binding to nucleic acid from cells of the tumour does not
match probe binding to nucleic acid from normal cells.
This may facilitate identification of appropriate
therapy, for example involving manipulation of MAP kinase
phosphatase activity in tumour cells (for which see
below).
As primers, oligonucleotides may be used in PCR with
cDNA derived from mRNA isolated from any tissue of human
or animal or plant or microbial origin to amplify related
genes which are likely to act as MAP kinase phosphatases.
In addition genomic DNA may also be amplified providing a
method of accessing all MAP kinase related genes in the
genome of, e.g. the human, mouse etc. The clones and
fragments already isolated may be used to isolate further
members of the gene family by low stringency
hybridisation. Preliminary experiments may be performed
by hybridising under low stringency conditions various
WO95/21923 2 1 ~ 2 9 6 7 PCT/GB95/00272
24
probes to Southern blots of human DNA digested with
restriction enzymes. Suitable conditions would be
achieved when a large number of hybridising fragments
were obtained while the background hybridisation was low.
Using these conditions cDNA libraries representative of
expressed sequences in various human or ~n ~1 or plant
tissues or libraries made from genomic DNA of human or
~n; ~1 or plant or microbial origin. The screening of
genomic libraries by this method may lead to the
isolation of all such homologous genes from the above
species.
Where a full-length phosphatase encoding nucleic
acid molecule has not been obtained, a smaller molecule
representing part of the full molecule, such as those
shown in Figure 2, may be used to obtain full-length
clones. Inserts may be prepared from partial cDNA clones
and used to screen cDNA libraries made from any of
various human tissues. The full-length clones isolated
may be subcloned into m~mm~l ian expression vectors and
MAP kinase phosphatase activity assayed by co-
transfection into COS cells, or other suitable host
cells, with a reporter plasmid encoding MAP kinase or
other substrate, or a suitable fragment thereof. MAP
kinase has a different electrophoretic mobility depending
on whether it is phosphorylated or not. The shift
between the phosphorylated form to the dephosphorylated
form in the presence of active MAP kinase phosphatase is
detectable, eg using Western blotting.
WOgS/21923 ~ rS ~ e 2 1 82967 PCT/GB95/00272
For instance, the MAP kinase of the reporter plasmid
may be tagged, eg with a myc-epitope (32), which allows
detection using an anti-tag antibody. A suitable anti-
myc antibody is 9El0 (43). Many ways of labelling
proteins for detection/visua~isation are known to those
skilled in the art, so details need not be given here.
Indeed, anti-MAP kinase antibodies may be used, needing
no label to be added to the protein. COS cells co-
transfected with a labelled MAP kinase may be stimulated
with EGF, which, in the absence of phosphatase activity,
leads to a mobility shift in the MAP kinase which can be
detected by means of the label. The presence of MAP
kinase phosphatase activity within the cell leads to
abolition of the mobility shift, thus providing a
convenient assay for MAP kinase phosphatase activity, and
enabling identification of encoding nucleic acid.
Other assays for MAP kinase phosphatase activity of
a protein encoded by cloned nucleic acid may be used.
These include expression in a bacterial host, such as E.
coli, with a suitable tag or other label (eg a histidine
tag or as glutathione-S-transferase fusion protein),
followed by purification of the recombinant protein and
subsequent incubation with recombinant phosphorylated MAP
kinase. Dephosphorylation can be assayed by
scintillation counting. Of course, recombinant
production and purification of a MAP kinase phosphates
for mixing with MAP kinase may be by any technique known
in the art.
WO95/21923 2 1 8 2 9 ~ 7 PCT/GB95/00272
A reticulocyte lysate in vitro translation system
containing MAP kinase may be used, again to assay
phosphorylation of the MAP kinase by mobility shift.
Candidate phosphatase clones may be translated in vitro
in reticulocyte lysate and abolition of the mobility
shift assayed.
The mobility shift may be used in the screening of
effector molecules (activators or inhibitors) for a MAP
kinase phosphatase, in any of the above assay systems.
For instance, an inhibitor of the MAP kinase phosphatase
chosen for study will abolish or reduce the
dephosphorylating activity of the phosphatase and so
restore the mobility shift of MAP kinase or other
substrate, where appropriate. Such screens are provided
as an aspect of the present invention. Biochemical
assays may be used to screen for effector molecules.
In any assay, a suitable fragment of MAP kinase (or
any other substrate of the phosphatase of interest) may
be use, eg a fragment including a site of action of the
phosphatase.
A yeast two-hybrid system (43,44) may be used to
identify molecules that interact with a phosphatase
molecule. This system utilises a yeast containing a GAL4
responsive promoter linked to ~-galactosidase gene and to
a gene (His3) that allows the yeast to grow in the
absence of the amino acid histidine and to grow in the
presence of the toxic compound 3-aminotriazole. The
phosphatases may be cloned into yeast vectors that will
W095/21923 ~ PCT/GB95/00272
express these proteins as fusions with the DNA binding
domain of GAL4. These yeast may then be transformed with
cDNA libraries constructed from various human tissues in
vectors designed to express proteins as GAL4 activator
fusions. Yeast that have a blue colour on indicator
plates (due to activation of ~-galactosidase) and will
grow in the absence of histidine (and the presence of 3-
aminotriazole) may be selected and the library plasmid
isolated. The library plasmid may encode a protein that
can interact with the phosphatase. Such a protein may be
a molecule which interacts with the phosphatase and
modulates the activity. It also seems likely that
substrates for the phosphatase other than MAP kinase may
be isolated. These may be known non-classical (i.e. non
erk-type) MAP kinases or novel molecules involved in
signal transduction.
The provision of MAP kinase phosphatases and
encoding nucleic acid sequences therefor enables effector
molecules to be screened using a novel yeast system, eg
in Schizosaccharomyces pombe.
A phosphatase may be incorporated into the screening
system that has been devised for the identification of
molecules that can specifically inhibit components of the
MAP kinase system (46 and W094/23039). In this system
m~mm~l ian c-raf and MAP kinase have been introduced into
the yeast S. pombe so that they can complement the
sterility of yeast mutant in the Byrl or Byr2 genes.
These yeast genes are involved in the mating pathway.
WOgS/21923 2 ~ 8 2 ~ 6 ~ PCT/GB95/00272
28
The m~mm~l ian genes are able to substitute for components
of the pathway and can then be targeted. This screen may
be adapted so that the activity of the m~mm~l ian enzymes
(c-raf and MAP kinase) leads via activation of m~mm~l ian
MAP kinase to the production of ~-galactosidase enzyme so
that these yeast will be blue on suitable indicator
media. This system may be used to assay for substances
that can specifically inhibit the m~mm~l ian signal
transduction molecules by scoring for yeast that ha~e
lost their blue colour. The specificity of the screen is
ensured as any non-specific kinase or other inhibitors
would prevent growth of the yeast rather than simply loss
of the blue colour. Other markers, such as the mating
ability of the S. pombe strain, may be used.
Any MAP kinase phosphatase, including those provided
herein and the known CL100, may be incorporated into this
screening system. For instance, constitutive expression
of a phosphatase may be manipulated by the use of
suitable expression vectors to reduce partially the
activity of the m~mm~l ian MAP kinase present in the yeast
so that the ~-galactosidase activity is partially
reduced, resulting in a diminution of the blue colour of
the yeast on suitable indicator plates. This system may
then be used to screen for compounds that can inhibit
(leading to a stronger blue colour) or activate
(attenuating the blue colour) a chosen phosphatase. Such
compounds may be useful therapeutic agents, for example
antiproliferative or anti-inflammatory drugs.
WO95/21923 ~ 2 1 8 2 9 ~ 7 pcTlGs9sloo272
It is well known that pharmaceutical research
leading to the identification of a new drug generally
involves the screening of very large numbers of candidate
substances, both before, and even after, a lead compound
has been found. This is one factor which makes
pharmaceutical research very expensive and time-
consuming, so that a method for assisting in the
screening process can have considerable commercial
importance.
Of course, the marker used may be a simple
"positive/negative" indicating the presence or absence of
MAP kinase activity and so the respective absence or
presence of MAP kinase phosphatase activity. The
inhibition of the MAP kinase phosphatase by a test
molecule (allowing MAP kinase activity) would then
manifest as a positive result, eg blue colour, mating
ability and so on, identifying the molecule as an
inhibitor of the phosphatase.
It seems probable that the MAP kinase phosphatases
act to switch off cellular proliferation. As such, loss
of their enzyme activity by e.g. deletion or mutation may
lead to uncontrolled cellular proliferation and cancer.
Several regions of the human genome have been described
which show loss of heterozygosity, i.e. are deleted in
various human tumours. Whether any of the phosphatase
genes provided herein map near any of these regions of
the human genome may be determined. Methods for the
mapping of genes within the human genome are well known
W~95/21923 ~ t 8 ~ 9 6 7 PCT/GB95/00272
and include analysis using specific PCR primers of the
presence of genes in cell hybrids segregating human
chromosomes as well as fluorescence in situ hybridisation
(FISH). Any genes which are deleted in specific tumours,
may be useful as reagents for the classification of such
tumours and their diagnosis.
STY8 like sequences have been detected on human
chromosomes lq and 8p using fluorescence in situ
hybridisation (FISH). More detailed mapping and analysis
of tumour material may be used to confirm tumour
suppressor function.
As discussed, methods of diagnosis, comprising the
use of any nucleic acid molecule with a sequence provided
herein, or any fragment, mutant, allele or derivative
thereof, are encompassed by the present invention.
Conveniently, this may involve use of specific PCR
primers that recognise polymorphic regions of these genes
or by using probes derived from these genes on Southern
blots of DNA isolated from tumour material.
Also provided by the present invention are
therapeutic methods employing MAP kinase phosphatase
polypeptides, antibodies thereto or encoding nucleic acid
therefor.
In principle, gene therapy using nucleic acid
encoding a polypeptide with MAP kinase phosphatase
activity may be used in treatment of any disorder which
arises from a loss of wild-type MAP kinase phosphatase
activity. Such disorders will generally be cell-
WO 9S/21923 `. ~; ? .~ 2 1 8 2 9 6 7 PCT/GD95/~,~272
31
proliferative, involving inappropriate cell growth as aresult of cellular pathways which normally regulate
growth using a MAP kinase being "switched on" with the
"off-switch", dephosphorylation of MAP kinase by a MAP
kinase phosphatase, not being applied as normal.
Disorders of cell proliferation and growth may be benign
or malignant.
Retinoblastoma is the "classic" example of a cell
proliferative disorder which results from loss of
function of a normally expressed gene. Individuals who
are heterozygous for the Rb-l gene, i.e. they have only
one wild-type copy of the gene, are predisposed to get
the disease because of the increased likelihood of a
mutation leaving them with no working copy of the gene.
Gene therapy for retinoblastoma, using a nucleic acid
construct comprising the Rb-l gene, has been proposed
(e.g. in WO91/15580 and WO94/06910). Introduction of the
Rb-l gene into a tumour cell that has lost the Rb-l gene
results in suppression of growth of the tumour.
Significantly, although the tumorigenic phenotype was
suppressed by the Rb-l gene, it had no effect on normal
cells.
p53 is another gene which has a "tumour suppressor
function" and is an appropriate target for gene therapy.
In accordance with the present invention, gene
therapy may be employed in the treatment of a disorder,
especially a disorder of cell proliferation, involving
loss of activity, total or partial, of a MAP kinase
WO9S/21923 PCT/GB9S/00272
2 ~ 6 7
phosphatase. A method of treatment practised on the
human or ~n;m~l body in accordance with the present
invention may comprise administration of nucleic acid
encoding a polypeptide which has MAP kinase phosphatase
activity, as disclosed herein. Preferably, the nucleic
acid forms part of a gene construct enabling expression
within cells of the individual. Conveniently, the
nucleic acid may be introduced into cells using a
retroviral vector, preferably one which will not
transform non-proliferating cells, or using liposome
technology.
The treatment may be of existing disease, or it may
be prophylactic. Preventative treatment may be
appropriate for individuals who have been identified as
at risk of developing a disorder, e.g. because they lack
two copies of a wild-type MAP kinase phosphatase gene or
have mutated genes encoding a MAP kinase phosphatase with
aberrant activity.
A~m;n;stration is preferably in a "therapeutically
effective amount", this being sufficient to show benefit
to a patient. The actual amount administered, and rate
and time-course of administration, will depend on the
nature and severity of what is being treated.
Prescription of treatment, eg decisions on dosage etc, is
within the responsibility of general practioners and
other medical doctors. A~m'n;stration may be alone or in
combination with other treatments, either simultaneously
or sequentially dependent upon the condition to be
2 1 ~2967
WO95/21923 PCT/GB95/00272
~,; ~, ~; . , !
33
treated.
Pharmaceutical compositions for administration in
accordance with the present invention may comprise a
pharmaceutically acceptable excipient, carrier, buffer,
stabiliser or other materials well known to those skilled
in the art. Such materials should be non-toxic and
should not interfere with the efficacy of the active
ingredient. The precise nature of the carrier or other
material will depend on the route of administration,
which may be in principle be oral, intranasal, topical,
or by cutaneous, subcutaneous, intravenous or
intramuscular injection.
Pharmaceutical compositions for oral administration
may be in tablet, capsule, powder or liquid form. A
tablet may comprise a solid carrier such as gelatin or an
adjuvant. Liquid pharmaceutical compositions generally
comprise a liquid carrier such as water, petroleum,
animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other
saccharide solution or glycols such as ethylene glycol,
propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous
injection, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and
stability. Those of relevant skill in the art are well
able to prepare suitable solutions using, for example,
isotonic vehicles such as Sodium Chloride Injection,
Wo95/21923 2 1 8 2 9 ~ 7 PCT/~b5J~ 72
Ringer's Injection, Lactated Ringer's Injection.
Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
Injection may be used to deliver nucleic acid to
disease sites, such as tumours. Internally, e.g. in
internal organs, body cavities etc., suitable imaging
devices may be employed to guide an injecting needle to
the desired site.
In some proliferative diseases, such as those of the
bone marrow, leukaemias etc., it may be desirable in
certain cases to remove cells, including normal and
tumour cells, from the body, treat them, then return them
to the body.
Nucleic acid may be introduced locally into cells
lS using transfection, electroporation, microinjection,
lipsomes, lipofectin or as naked DNA or RNA, or using any
other suitable technique. Retroviral vectors (Wilson et
al., PNAS USA, 85 : 3014, Gilboa (1982) J. Virology 44 :
845 and Hocke (1986) Nature 320:275) and vaccinia viruses
(Chakrabarty et al (1985) MOl. Cell Biol. 5: 3403) are
amongst the choices available to those skilled in the
art. Proliferating cells may be targeted especially
using defective retroviruses lacking genes required for
replication, since such retroviruses must rely on
25 cellular DNA replication for integration of their genome
and expression of polypeptides encoded therein. Such
~replication incompetent" retrovirus vectors include
those described by Chen et al (Science 250: 1576-80,
WO9S/21923 ~ 2 ~ 8 2 9 6 7 PCT/~b5SIW272
1990) and Miller et al (BioTechni~ues 7: 980-990, 1989).
According to further aspects of the present
invention there are provided a pharmaceutical
composition, as disclosed, comprising nucleic acid
encoding a polypeptide with MAP kinase phosphatase
activity, as disclosed, for use in therapy or
prophylaxis, especially of a cell-proliferative disorder,
and the use of nucleic acid encoding a polypeptide which
has MAP kinase phosphatase activity in the manufacture of
a composition or medicament for use in treatment as
disclosed.
Techniques of introduction of nucleic acid into
m~mm~l ian cells may also be used to create transgenic
animals, e.g. rodents such as mice, rats, hamsters etc.,
which carry one or more genes encoding defective, or at
least altered, MAP kinase phosphatase. For instance,
techniques involving homologous recombination may be
used. The ~n;m~l S may have a germline or somatic mutated
gene encoding a polypeptide with MAP kinase phosphatase
activity. Such transgenic ~n; m~l S may be used in
studying progression of disease involving a MAP kinase
phosphatase or in screening or assessment of substances
for therapeutic action, or testing of therapies, for
example involving gene therapy wherein a wild-type gene
may be introduced.
Aspects of the present invention will now be
illustrated with reference to the accompanying figures,
WOgS/21923 2 ~ 8 ~ 9 ~ 7 PcTlGs9sloo272
by way of example and not limitation. Further aspects
and embodiments will be apparent to those of ordinary
skill in the art. All documents mentioned in the text
are incorporated herein by reference.
In the figures:
Figure l is a schematic diagram of the m~mm~l ian MAP
kinase signal transduction pathway (from ref 2). Ligand
interacts with a receptor tyrosine kinase at the cell
surface. The receptor becomes phosphorylated on tyrosine
residues which allows it to associate with GRB2 and the
ras exchange factor SOS. SOS activates ras to the GTP
bound form which is then able to interact with the MAP
kinase kinase kinase raf. In a process which is not yet
understood this activates raf so that it can
phosphorylate MAP kinase kinase, which in turn
phosphorylates MAP kinase on threonine and tyrosine
residues. This activated form of MAP kinase can then
stimulate cellular profileration.
Figure 2 shows DNA sequences of novel phosphatase
molecules. STY2-STY4 are PCR products amplified from RNA
produced from A431 cells as described in the text. STY 5
and STY6 were isolated by screening a hamster liver cDNA
library with a mixture of STY2 and STY3 probes shown in
part (a) and (b). STY7-STYlO are parts of cDNA clones
isolated by screening a human brain cDNA library with a
mixture of STY2 and STY3 probes shown in part (a) and
(b). All sequences apart from STY7 and STYlO show
~ ~ . r 2 1 82967
WOgS/21923 ~ PCT/~b5S~W27
37
homology to CL100. In the case of these clones the
sequence shown does not show homology to CL100 but the
- cDNA clones hybridised strongly to the STY2/3 probe
suggesting that these clones also encode novel
phosphatase genes. (a) - STY2; (b) - STY3; (c) - STY4;
(d) - STY5; (e) - STY6; (f) - STY7; (g) - STY8; (h) STY9;
(i) STY10.
Figure 3 shows deduced amino acid sequences of
phosphatase clones aligned with the amino acid sequence
of CL100: (a) - STY2, STY3, STY 4 AND STY5; (b) STY6;
(c) STY 9; (d) STY 8. For parts (a) - (c) spaces
indicate residues that are identical with CL100 and dots
indicate residues which have not yet been determined.
For part (d) which is a comparison of the full length
clone for STY8 with CL100 dashes (-) indicate gaps
introduced into the sequences to optimise their
alignment. Shaded residues correspond to residues that
are identical between STY8 and CL100.
The amino acid sequences shown correspond to
residues 177-255 for part (a), 231-302 for part (b). 223-
267 for part (c) and 1-367 for part (d).
Figure 4 shows proof that STY8 encodes MAP kinase
phosphatase activity. Protein extracts were prepared
from COS cells transfected with various recombinant
plasmids before or after stimulation of the cells with
EGF. These extracts were electrophoresed on
SDS/polyacrylamide gels and the proteins then transferred
to a nitrocellulose membrane. This membrane was then
~ l 8296 7
WO95/21923 PCT/GB95100272
incubated with the anti-myc antibody 9ElO, treated by the
ECL procedure and the resulting chemiluminescence
detected on x-ray film. It can be seen that in the
absence of stimulatory ligand tEGF) the anti-myc antibody
9E10 reveals only a single band of MAP kinase on western
blotting (lane 1). In the presence of EGF (lane 2) a
clear doublet of bands is present indicating the partial
phosphorylation of the MAP kinase. This is unaffected by
expression of the parental expression vector (lanes 3 and
4). However, expression of CL100 or STY8 in the presence
of EGF (lanes 7-10) leads to abolition of the EGF induced
shift indicating that both these molecules encode MAP
kinase phosphatases. Lanes 5 and 6 in which the cells
are transfected with Myc-tagged STY8 shows that the STY8
protein is indeed expressed. Lane 1 - MAPK; Lane 2 -
MAPK + EGF; Lane 3 - MAPK + pMT; Lane 4 - MAPK + pMT +
EGF; Lane 5 - Myc-STY8; Lane 6 - Myc-STY8 + EGF; Lane 7 -
MAPK + CL100; Lane 8 - MAPK + CL100 + EGF; Lane 9 - MAPK
+ STY8; Lane 10 MAPK + STY8 + EGF.
Figure 5 shows the results of autoradiography of
SDS-polyacrylamide gels on which samples containing (a)
MAP kinase kinase and ERK2 or (b) MAP kinase kinase, ERK2
and STY8 were incubated in the presence of 32P-ATP for
times indicated in minutes across the top of the lanes.
The positions of phosphorylated ERK2 and STY8 are
indicated. The left hand lane shows molecular weight
markers.
`` 21 82967
WO95/21923 PcT/Gsgs/00272
}:
Isolation of MAP kinase phosphatase encoding genes
To identify related protein amino acids sequences
human CL100 and its murine homologue 3CH134 and the human
PAC-1 gene [42], a related T cell specific gene of
unknown function, were compared. It proved possible to
design degenerate PCR primers, baæed on conserved regions
of the proteins. These primers were used to amplify
related sequences from cDNA made from poly(A)~RNA isolated
from the hllm~n squamous cell line A431. A fragment of
270bp was purified and subcloned. Of fifty individual
clones sequences six proved to be identical to CL100. A
further twelve clones were found to be homologous to, but
distinguishable from, CL100:- STY2 isolated six times and
STY3 four times, with single isolates of STY4 and STY5.
In order to identify further related genes, we screened
human brain and liver cDNA libraries with a mixed probe
from STY2 and-3 PCR products. Several hybridising clones
were analysed in more detail by restriction endonuclease
mapping and partial DNA sequencing. This resulted in the
identification of several additional gene families, STY6-
10, with STY1 being CL100. In total nine new genes were
identified and these are compared to amino acid sequences
of CL100, see figure 3. The high degree of similarity of
- these genes suggested that they encode proteins with MAP
kinase phosphatase activity.
Cel 1 Cul ture and RNA Prepara ti on
A431 cells were grown in Dulbecco's modification of
~ 1 82967
WO95/21923 PCT/GB9Sl00272
Eagle's ~;n;m~l essential medium (DMEM) supplemented with
10~ fetal calf serum. Total cellular RNA was prepared
with RNAzolB(Promega) and poly(A)+RNA isolated with
Dynabeads oligo(dT)25(Dynal).
Isolation of CL100-related cDNAs
Two degenerate oligonucleotides
TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A)CC(A,T)GT(A,G,T)
GA and AT(G,C,T)CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC
were designed based on amino acid sequences, YDQGGPVE and
VHCQAGI conserved between human and mouse CL100 and the
human PAC-1 gene. A431 poly (A)- RNA (l~g) was reverse
transcribed with SuperScript reverse transcriptase (BRL-
GIBCO) and subject to PCR on a Techne PHC-1 thermal
cycler with these oligonucleotides (47) under the
following conditions : 94C for 30sec, 50C, 30sec, 72C 1
min. A 270bp band was purified by agarose gel
electrophoresis and subcloned into pBluescript.
Fifty individual subclones were sequenced and of
these six proved to be CL100. Twelve others were found
to be homologous to but not identical to CL100, and these
were grouped as four different potential phosphatases,
designated STY2 STY5 with CL100 being STY1. STY6-STY10
were isolated by screening cDNA-libraries with a 32P-
labelled probe made from the inserts of plasmidscontaining STY2 and STY3 sequence shown in Figure 2a.
STY6 was isolated from a human brain library.
- 2 1 82967
WO95/21923 PCT/GB95/00272
Structural Analysis of STY cDNAs
One of the cDNA clones isolated from the human brain
is full length. Colinear alignments of the STY genes
with CL100 show that amino acids around the highly
conserved catalytic domain differ, and two conserved
regions between CL100 and cdc25 are also present in STY8.
Studies on the genomic structure of 3CH134 reveal that
the transcription unit is 2.8kbp long and split into four
exons [46]. It will be of interest to elucidate the
genomic structure of the STY genes, and determine if
their promoter regions contain consensus sequences for
transcription factors. Prel; m; n~ry studies suggest that
STY8 has a similar gene structure to 3CH134.
Functional Assays
The human CL100 and its murine counterpart 3CH134
function as immediate-early genes whose transcription is
rapidly and transiently induced within minutes, with
protein accumulation seen in the first hour upon growth
factor stimulation [7,48]. As observed for the
expression of several ; mm~A; ate-early genes, the rapid
increase in growth factor receptor tyrosine kinase
activity and subsequent activation of signalling
- molecules needs to return to normal levels to avoid
abnormal growth. One method for accomplishing this
implicates protein phosphatases whose expression is
induced by external signals, such that they are present
in the cell only under certain circumstances.
WO95/21923 2 1 8 2 9 6 7 pcTlGs95loo272
42
Evidence indicates that when CLlO0 and 3CHl34 are
expressed in vitro [4,5] or in vivo [7], the gene product
leads to selective dephosphorylation of p42~Pk blocking
its activation by serum, oncogenic Ras, or activated Raf,
whilst the catalytically inactive mutant of the
phosphatase augments MAP kinase phosphorylation.
We tested whether the phosphatase STY8 exhibited
similar specificity in vivo using a COS cell transient
expression system. We cotransfected Cos cells with the
reporter plasmid pEXV3-Myc-p42~Pk together with various
plasmids including pMT-Myc-STY8. Figure 4 is typical of
such an experiment.
It can be seen that in the absence of stimulatory
ligand (EGF) the anti-myc antibody 9ElO reveals only a
single band of MAP kinase on western blotting (lane l).
In the presence of EGF (lane 2) a clear doublet of bands
is present indicating the partial phosphorylation of the
MAP kinase. This is unaffected by expression of the
parental expression vector (lanes 3 and 4). However
expression of CLlO0 or STY8 in the presence of EGF (lanes
7-lO) leads to abolition of the EGF induced shift
indicating that both these molecules encode MAP kinase
phosphatases. Lanes 5 and 6 in which the cells are
transfected with Myc-tagged STY8 shows that the STY8
protein is indeed expressed.
We have demonstrated that recombinant STY8 can
dephosphorylate MAP kinase (erk2) when the two proteins
are incubated in vitro. Recombinant activated MAP kinase
Wo95/2l923 ; - 2 1 8 2 9 6 7 PCT/~b55~^272
kinase (MEK/EE) (50ng) was incubated with recombinant
ERK2 (3~g) or recombinant ERK2 (3~g) and recombinant STY8
(0.75~g) in the presence of 32P-ATP (2.5nCi) in 50mM Tris-
Cl (pH 7.5)/O.lmM EGTA/lOmM MgAcetate/0.125mM ATP for the
times indicated (in minutes) in Figure 5. Samples were
then electrophoresed on a 10~ SDS/polyacrylamide gel and
the gel dried and autoradiographed. The positions of
phosphorylated ERK2 and ~surprisingly) STY8 are shown in
the Figure.
STY8 is shown by this experiment surprisingly to be
itself a substrate of the MAP kinase erk2. This can be
seen in Figure 5 by the appearance of the phosphorylated
ST8 during the incubation with erk2. The significance of
this phosphorylation is at present unknown but several
potential sites for phosphorylation are located in the
sequence of STY8. It is possible that phosphorylation of
STY8 by erk2 serves to modulate its activity, either
increasing or reducing it. Another possibility is that
phosphorylation targets the protein for degradation. A
further possibility is that the presence of potential MAP
kinase phosphorylation sites within STY8 serves to
facilitate the interaction of erk2 and STY8 and the
consequence is phosphorylation of STY8 and
dephosphorylation of erk2.
STY8 has also been cloned into a baculovirus
transfer vector, resulting, after recombination, in
production of a Glutathione-S-transferase (GST) - STY8
fusion protein. The vector was co-transfected into
WO95/21923 2 ! `~ 2 9 ~ 7 PCT/~b~'/00272
insect cells along with linear viral genomic DNA.
Recombination resulted in production of viral particles
containing baculovirus DNA having incorporated the
expression vector. This virus was used to infect further
insect cells and the recombinant GST-STY8 protein
purified by affinity chromatography on glutathione
agarose.
STY2 has been fully cloned and seguenced and shown
to have MAP kinase phosphatase activity, i.e. the ability
the dephosphorylate a MAP kinase (ERK2) (52). The
sequence is related to CLlO0 and STY8 throughout,
particularly in the catalytic domain.
The provision of the nucleic acid encoding MAP
kinase phosphatases enables the following to be
performed.
E~pressi on Pa t terns of STY cDNAs
Experiments have demonstrated that 3CHl34 is
expressed predominantly in the lung of the adult mouse
[4]. We have shown through northern blot analysis that
STY8/9 and CLlO0 are expressed ubiquitously across a
range of tissues. Interestingly, the expression of
3CH134 corresponds to post-mitotic cells [48], which
suggests the phosphatase may play a role in cellular
differentiation, acting as a negative effector of cell
growth. We have also shown through microinjection that
- 2 1 82967
woss/21923 : - PCT/GB95/00272
Myc-tagged STY8 is located in the nucleus of transfected
cells. This is where a MAPK phosphatase might be
expected to act in the light of evidence that reports MAP
kinase activity is biphasic, with the second sustained
peak correlating with nuclear translocation and
initiation of DNA synthesis [49].
The MAP kinase pathway promotes cell proliferation
and tumorigenesis, and so MAP kinase phosphatases may
function as tumour suppressor genes. We intend to
determine the chromosomal location of the STY genes using
a chromosomal hybrid panel of hamster cells with single
human chromosomes. These will be screened by PCR using
unique primers to each of the STY genes, and the exact
position identified using in situ hybridisation. (STY8
like sequences have been detected using flurescence in
situ hybridisation on human chromosomes lq and 8p.)
These loci may then be compared with regions of known
loss of heterozygosity in the BICR cell lines or that
correspond to known tumour suppressor loci.
wog5/21s23 2 8 2 9 6 7 PCT/GB95/00272
46
References
1. Noguchi, T. et al., Mol. Cell. Biol. 13, 5195-5205
(1993).
2. Crews, C.M., Alessandrini, A. & Erikson, R.L. Proc.
Natl. Acad. Sci. USA
88, 8845-8849 (1991).
3. Marshall, C.J. Current Opinions in Genetics and Dev.
(1994).
4. Keyse, S.M. & Emslie, E.A. Nature 359, 644-647
(1992).
5. Charles, C.H., Abler, A.S. & Lau, L.F. Oncogene 7,
187-190 (1992).
6. Alessi, D.R., Smythe, C. & Keyse, S.M. Oncogene 8,
2015-2020 (1993).
7. Charles, C.H., Sun, H., Lau, L.F. & Tonks, N.K.
Proc. Natl. Acad. Sci. 90,
5292-5296 (1993).
8. Sun, H., Charles, C.H., Lau, L.F. & Tonks, N.K. Cell
75, 487-493 (1993).
9. Ullrich, A. & Schlessinger, J. Cell 203-212 (1990).
10. Pawson, T. Current Opinions in Genetics and Dev. 2,
4-12 (1992).
11. Brautigan, D.L. Biochim. Biophys. Acta 1114, 63-77
(1992).5 12. William-Lau, K.H. & Baylink, D.J. Current Opinions
in Oncogenesis 4, 451-
471 (1993).
13. Walton, K. & Dixon, J.E. Ann. Rev. Biochem. 62, 101-
2 1 8 2 9 6 7
WO95/21923 PCTIGB95/00272
120 (1992).
14. Woodford-Thomas et al., J. Cell Biol. 117, 401-414
(1992).
15. Cook, D.E. et al., Proc. Natl. Acad. Sci. USA 87,
7280-7284 (1990).
16. Pallen, C.J. & Tong, P.H. Proc. Natl. Acad. Sci. USA
88, 6996-7000 (1991).
17. Gruppuso, P. et al., J. Biol. Chem. 266, 3444-3448
( 1991 ) .
18. Yi, T. et al., Mol. Cell. Biol. 13, 7577-7586
(1993).
19. David, M. et al., Mol. Cell. Biol. 13, 7515-7521
(1993).
20. Tojo, A. et al., Exp. Cell. Res. 171, 16-23 (1987).
21. Klarlund, J.K. Cell 707-717 (1985).
22. Brown-Schimer, S. Cancer Research 52, 478-482
(1992).
23. Ramponi, P. Int J. Cancer 51, 652-656 (1992).
24. Zhang, X.M. Nature 359, 336-339 (1992).
25. Boulton, T.G., et al. Cell 65, 663-675 (199lb).
26. Anderson, N.G., Maller, J.L., Tonks, N. & Sturgill,
T.W. Nature 343, 651-
653 (1990).
27. Payne, D.M., et al. EMBO J. 10, 885-892 (1991).
28. Margolis, B. Cell Growth and Diff. 3, 73-88 (1992).
29. Chardin, P. Science 260, 1338-1343 (1993).
30. Buday, L. & Downward, J. Cell 73, 611-620 (1993).
31. de Vries Smits, A.M.M., Burgering, B.M.T., Leevers,
WOgS/21923 2 1 ~ 2 9 6 7 PCT/~b93J~v27~
48
S.J., Marshall, C.J. &
Bos, J.L. Nature 357, 602-604 (1992).
32. Leevers, S.J. & Marshall, C.J. EMBO J. 11, 569-574
(1992).
33. Howe, L.R., et al. Cell 71, 335-342 (1992).
34. Kyriakis, J., et al. Nature 358, 417-21 (1992).
35. Dent, P., et al. Science 257, 1404-1407 (1992).
36. Van Aelst, L., Barr, M., Marcus, S., Polverino, A. &
Wigler, M.
Proc.Natl.Acad.Sci. 90, 6213-6217 (1993).
37. Warne, P., Viciana, P. & Downward, J. Nature 364,
352-355 (1993).
38. Lange-Carter, C., Pleiman, C., Gardner, A., Blumer,
K. & Johnson, G. Science
260, 315-319 (1993).
39. Posada, J. & Cooper, J.A. Science 255, 212-215
(1992).
40. Nebreda, A.R., Porras, A. & Santos, E. Oncogene 8,
467-477 (1993).
41. Gomez, N. & Cohen, P. Nature 353, 170-173 (1991).
42. Rohan, P.J., et al. Science 259, 1763-1766 (1993).
43. E~an, G.I., Lewis, G.K., Ramsay, G. & Bishop, J.M.
Mol. Cell. Biol. 5, 3610-
3616 (1985).
44. Fields, S. & Song, O.K. Nature 340, 245-246 (1989).
45. Durfee, T., et al. Genes Dev. 7, 555-569 (1993).
46. Hughes, D., Ashworth, A. & Marshall, C. Nature 364,
349-352 (1993).
2 1 82967
WO95/21923 ~ CT/GB95/00272
49
47. Ashworth, A. in Transcription Factors : A Practical
Approach" (Latchman,
D., ed.) pp 125-142. IRL Press, Oxford, UK. (1993).
48. Noguchi, T. et al., Mol. Cell. Biol. 13, 5195-5205
(1993).
49. Traverse, S., Gomez, N., Paterson, H., Marshall, C.
~ Cohen, P. Biochem. J.
288, 351-355 (1992).
50. Da~is, R.J. Trends in Biochem. Sci. 19, 470-473
(1994).
51. Sun, H., Tonk, N. and Bar-Sagi, D. Science 266, 285-
288 (1994).
52. ISh;h~s-h;~ T., Bottaro, D.P., Michieli, P., Kelley,
C.A. and Aaronson, S.A. J.
Biol. Chem. 269, 29897-29902 (1994).
