Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SUBSTRATE TRAPPING PROTEIN TYROSINE PHOSPHATASES
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-in-Part ofU.S. Application Serial No.
09/144,925, filed September 1, 1998, which is a Divisional of U.S. Application
No. 08/685,992, filed July 25, 1996. This application also claims the benefit
of U.S.
Provisional Application No. 60/137,319 (attorney docket number CSHL99-06P),
filed
June 3, 1999. The teachings of these applications are incorporated herein by
reference
in their entirety
STATEMENT OF GOVERNMENT INTEREST
Work described herein was supported by government funding under
research grant CA 53840 awarded by the National Institutes ofHealth. The
government
may have certain rights in this invention.
TECHNICAL FIELD
The present invention relates generally to compositions and methods
useful for treating conditions associated with defects in cellular biochemical
pathways
such as those controlling cell proliferation, cell differentiation and/or cell
survival. The
invention is more particularly related to substrate trapping mutants of
protein tyrosine
phosphatase polypeptides, and variants thereof. The present invention is also
related
to the use of such polypeptides to identify antibodies and other agents,
including small
molecules, that modulate biological signal transduction and cellular
biochemical
pathways.
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B~CKGROLT1D OF THE INVE~T1DN
Reversible protein tyrosine phosphorylation. coordinated by the action of
protein tyrosine kinases (PTKs) that phosphorylate certain tyrosine residues
in
polypeptides. and protein tyrosine phosphatases (PTPs) that dephosphorylate
certain
phosphotyrosine residues, is a kev met:~hanism in regulating manv cellular
activities. It
is becoming apparent that the diversity and complexity of the PTPs and P'TKs
are
comparable. and that PTPs are equally important in delivering both positive
and
negative signals for proper function of cellular machinery. Regulated tyrosine
phosphorylation contributes to specific pathways for biological signal
transduction.
including those associated with cell division, proliferation and
differentiation. Defects
and/or malfunctions in these pathways may underlie certain disease conditions
for
which effective means for intervention remain elusive, including for example.
malignancy. autoimmune disorders, diabetes. obesity and infection.
The protein tyrosine phosphatase (PTP) family of enzymes consists of
1 ~ more than X00 structurally diverse proteins that have in common the highly
conserved
250 amino acid PTP catalytic domain. but which display considerable variation
in their
non-catalytic se~nents (Charbonneau and Tonks. 1992 Annu. Rev. Cell Biol.
8:=~6~
493; Tonks. 1993 Semin. Cell Biol. x:373-X53). This structural diversity
presumably
reflects the diversity of physiological roles of individual PTP family
members. which in
certain cases have been demonstrated to have specific functions in ~owth.
development
and differentiation (Desai et al.. 1996 Cell 8~:~99-609; Kishihara et al..
1993 C~ll
~-~:1-~S-1~6; Perlcins et al.. 1992 Cell ~0:'??5-236; Pingel and Thomas. 1989
Cell
.18:10»-106; Schultz et x1..1993 Cell -3:1~~-1~5~).
a.~lthoush recent studies have also generated considerable information
regarding the structure. expression and regulation of PTPs. the nature of the
tyrosine
phosphorylated substrates throw which the PTPs exert their elects remains to
be
determined. Studies with a limited number of synthetic phosphopeptide
substrates have
demonstrated some differences in the substrate selectivities of different PTPs
(Cho et
al.. ; 99 ~ Protein Sc: l. ': 97 -98-~: Dec:~ert et al.. 199: Eur. J. Biochem.
?~ I :67~-681 ~.
.-W alvses of PTP-mediated dephosphoryiation of PTP substrates suggesmhat
catalytic
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activity may be favored by the presence of certain amino acid residues at
specific
positions in the substrate polypeptide relative to the phosphorylated tyrosine
residue
(Ruzzene 'et al.. 1993 Eur. J. Biochem. ?ll:'?89-?9~; Zhang et al.. 1994
Biochemistry
33:??85-~~90). Thus. although the phvsiolo~ical relevance of the substrates
used in
these studies is unclear. PTPs display a certain level of substrate
selectivity in vitro.
The PTP family of enzymes contains a common evolutionarily
conserved seu~nent of approximately 250 amino acids known as the PTP catalytic
domain. Within this conserved domain is a unique signature sequence motif.
[I/V]HC~G~[S/T~G SEQ ID N0:36,
that is invariant among all PTPs. The cysteine residue in this motif is
invariant in
members of the family and is known to be essential for catalysis of the
phosphoryrosine
dephosphorylation reaction. It functions as a nucleopbl1e to attack the
phosphate
moiety present on a phosphotyrosine residue of the incoming substrate. If the
cysteine
residue is altered by site-directed mutagenesis to serine (e.g.. in cysteine-
to-serine or
1 ~ ''CS"mutants) or alanine (e.g.. cysteine-to-alanine or 'CA'~ mutants). the
resulting PTP
is catalvtically attenuated but retains the ability to complex with. or bind.
its substrate,
at least in vitro. Such mutants can be made, for example, using the PTP family
member
MKP-1 (Sun et al.. 1993 Cell i.i:48?--X93), as well as other PTPs. However,
although
these CS mutants can in 'eneral bind erTectively to phosphoryrosyl substrates
in vitro to
?0 form stable enzyme-substrate complexes. in many cases such complexes cannot
be
isolated in vivo, for example when both the mutant PTP and the phosphoryrosyl
protein
substrate are present together within a cell. Thus. the CS mutants are of
limited
usefulness and cannot be employed to isolate all combinations of PTPs and
substrates.
Currently. desirable Goals for determining the molecular mechanisms
that Govern PTP-mediated cellular events include. inter alia. determination of
PTP
interacting molecules. substrates and binding parmers. and identification of
agents that
relate PTP activities. In some situations. however. current approaches may
lead to an
understandina_ of certain aspects of the regulation of tyrosine
phosphorylation by PTPs.
but still may not provide strategies to control specific tyrosine
phosphorylation and,~or
30 dephosphorylation events Within a cell.
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Accordingly. there is a need in the art for an improved ability to regulate
phosphotvrosine signaling, including regulation of PTPs. ~n increased
understanding
of PTP re_ulation may facilitate the development of methods for modulating the
activity of proteins involved in phosphoryrosine signaling pathways, and for
treating
conditions associated with such pathways. The present invention fulfills these
needs
and further provides other related advantages.
SUMMARY OF THE ITJVENTION
The present invention provides hove! substrate trapping mutant or altered
forms of mammalian PTPs, also referred to as substrate trapping PTPs (ST-
PTPs),
which bind (trap) one or more substrates of the PTP. Binding of the ST-PTP to
a PTP
substrate results in the formation of a complex that can be readily observed.
and. if
desired. isolated_ and characterized. These mutant forms of PTPs have
attenuated
catalytic activity (lack catalytic activity or have reduced catalytic
activity) relative to the
wild type PTP, but retain the ability to bind tyrosine phosphorylated
substrates) of the
I~ wild type PTP. ST-PTPs are useful, for example, to determine the fine
substrate
specificity of one or more PTPs.
It is an aspect of the invention to provide a substrate trapping mutant
protein tyrosine phosphatase in which the wildtype protein tyrosine
phosphatase
catalytic domain invariant aspartate residue is replaced with an amino acid
which does
not cause si~ificant alteration of the Km of the enzyme but which results in a
reduction
in Kcat to less than I per minute: and at least one wildtype tyrosine residue
is replaced
with an amino acid that is not capable of being phosphorylated. In certain
embodiments
at least one wildtype tyrosine residue is replaced with an amino acid that is
alanine.
cysteine_ aspartic acid. ~lutamine. glutamic acid. phenylalanine. ~lycine.
histidine.
?5 isoleucine. lysine. leucine. methionine, aspara:~ne. proline. arginine,
valine or
tryptophan. In certain other embodiments at least one tyrosine residue that is
replaced
is located in a protein tyrosine phosphatase catalytic domain. In certain
embodiments at
least one t'-rosine residue that is replaced is located in a protein tyrosine
phosphatase
active site. and in certain other embodiments at least one tyrosine residue is
replaced
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with phenylalanine. In certain other embodiments at least one tyrosine residue
that is
replaced is a protein tyrosine phosphatase conserved residue. and in certain
further
embodiments the conserved residue corresponds to tyrosine at amino acid
position 676
in human PTPH1. In certain embodiments at least one tyrosine residue is
replaced with
an amino acid that stabilizes a comple:c comprising the protein tyrosine
phosphatase and
at least one substrate molecule. In certain embodiments the substrate trapping
mutt
comprises a mutated PTPH1, and in certain embodiments the substrate trapping
mutant
comprises a mutated protein tyrosine phosphatase that is PTP 1 B. PTP-PEST,
PTP~!,
uLKP-I, DEP-1. PTP~. PTPXl, PTPX10, SHP?, PTP-P~, PTP-~IEGI, LC-PTP, TC-
PTP, CD45, LAR or PTPHl. In certain embodiments the substrate trapping mutant
comprises a mutated PTP-PEST phosphatase in which the amino acid at position
231 is
replaced with a serine residue.
It is another aspect of the present invention to provide a method of
identifying a tyrosine phosphorylated protein which is a substrate of a
protein tyrosine
phosphatase, comprising the steps of combining a sample comprising at least
one
tyrosine phosphorylated protein with at least one substrate trapping mutant
protein
tyrosine phosphatase. in which (i) the wildtype protein tyrosine phosphatase
catalync
domain invariant aspartate residue is replaced with an amino acid which does
not cause
significant alteration of the Km of the enzyme but which results in a
reduction in Kcat
to less than I per minute, and (ii) at least one wildtvpe tyrosine residue is
replaced with
an amino acid that is not capable of being phosphorylated_ under conditions
and for a
time sufficient to permit formation of a comple:c between the tyrosine
phosphorylated
protein and the substrate napping mutt protein tyrosine phosphatase; and
determining
the presence or absence of a comple:c comprising the tyrosine phosphorylated
protein
?5 and the substrate trapping mutant protein tyrosine phosphatase. wherein the
presence of
the comple:c indicates that the tyrosine phosphoryiated protein is a substrate
of the
protein tyrosine phosphatase with which it forms a comple:c. In certain
embodiments
the substrate trapping mutant comprises a mutated protein tyrosine phosphatase
that is
PTP1B: PTP-PEST. PTP~!. VIKP-1_ DEP-I. PTPu. PTPYI. PTPX10. SHP'_'. PTP-PEZ.
;0 PTP-VlEGI. LC-PTP. TC-PTP. CD~~. L~R or PTPH1. In certain embodiments the
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6
sample comprises a cell that e:cpresses the tyrosine phosphorylated protein.
and in
certain further embodimenu the cell has been transfected with at 1 east one
nucleic acid
molecule encoding the substrate. In certain other embodiments at least one
substrate
trapping mutant protein tyrosine phosphatase is e:cpressed by a cell. and in
certain
~ further embodimenu the cell has been transfected with at least one nucleic
acid
molecule encoding the substrate trapping mutant protein tyrosine phosphatase.
In
certain other embodiments the sample comprises a cell that e:cpresses (l) the
tyrosine
phosphoryiated protein which is a substrate of the protein tyrosine
phosphatase, and (ii)
the substrate trapping mutant protein tyrosine phosphatase. In certain other
embodimenu the cell has been transfected with (l) at least one nucleic acid
encoding the
substrate. and (ii) at least one nucleic acid encoding the substrate trapping
mutant
protein tyrosine phosphatase. In certain other embodimenu the sample comprises
a cell
lysate containing at least one tyrosine phosphorylated protein. and in certain
funkier
embodimenu the cell lysate is derived from a cell transfected with at least
one nucleic
1 ~ acid encoding the tyrosine phosphorylated protein. In certain other
further
embodiments the cell lysate is derived from a cell ttansfected with at least
one nucleic
acid encoding a protein tyrosine kinase. In certain other embodimenu at least
one
substrate trapping mutant protein tyrosine phosphatase is present within a
cell lysate.
and in certain further embodimenu the cell lysate is derived from a cell
transfected with
at least one nucleic acid encoding the substrate trapping mutant protein
tyrosine
phosphatase. In other embodiments. the tyrosine phosphoryiated protein is
~JCP.
p130'u. the EGF receptor. p~10 bcr:abl. VIAP lanase. Shc (Tiganis et al., 1998
:~lol.
Cell. Biol. 18:162?-16~~) or the insulin receptor.
Turnips to another aspect_ the present invention provides a method of
~5 identifying an anent which alters the interaction between a protein
tyrosine phosphatase
and a tyrosine phosphoryiated protein which is a substrate of the protein
tyrosine
phosphatase. comprising contacting in the absence and in the presence of a
candidate
aQent_ a protein tyrosine phosphatase and a tyrosine phosphorylated protein
which is a
substrate of the protein tyrosine phosphatase under conditions and for a time
sufficient
~0 for detectable dephosphon~lation of the substrate to occur. wherein the
tyrosine
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7
phosphorylated protein which is a substrate of the protein tyrosine
phosphatase is
identified by (I) combining a sample comprising at least one tyrosine
phosphorylated
protein with at least one substrate trapping mutant protein tyrosine
phosphatase. in
which (l) the wildtype protein tyrosine phosphatase catalytic domain invariant
aspartate
residue is replaced with an amino acid which does not cause significant
alteration of the
Km of the enzyme but which results in a reduction in Kcat to less than 1 per
minute,
and (ii) at least one wildtype tyrosine residue is, replaced with an amino
acid that is not
capable of being phosphorylated. under conditions and for a time sufficient to
permit
formation of a complex between the tyrosine phosphorylated protein and the
substrate
trapping mutant protein tyrosine phosphatase: and (2) determining the presence
or
absence of a complex comprising the tyrosine phosphorylated protein and the
substrate
trapping mutant protein tyrosine phosphatase. wherein the presence of the
complex
indicates that the tyrosine phosphorylated protein is a substrate of the
protein tyrosine
phosphatase with which it forms a complex: and comparing the level of
1~ dephosphorylation of the substrate in the absence of the agent to the level
of
dephosphorylation of the substrate in the presence of the agent wherein a
difference in
the level of substrate dephosphorylation indicates the agent alters the
interaction
between the protein tyrosine phospharase and the substrate.
In another aspect the present invention provides a method of identifying
an agent which alters the interaction between a protein tyrosine phosphatase
and a
tyrosine phosphorylated protein which is a substrate of the protein tyrosine
phosphatase,
comprising contacting in the absence and in the presence of a candidate agent.
a protein
tyrosine phosphatase and a tyrosine phosphorylated protein which is a
substrate of the
protein tyrosine phosphatase under conditions and for a time su~cient to
permit
formation of a complex between the tyrosine phosphorylated protein and the
substrate
trappinmutant protein tyrosine phosphatase. wherein the substrate trapping
mutant
protein tyrosine phosphatase comprises a mutated protein tyrosine phosphatase
in which
(l) the wildtype protein tyrosine phosphatase catalvric domain invariant
aspartate
residue is replaced with an amino acid which does not cause si~ificant
alteration of the
Km of the enzyme but which results in a reduction in Kcat to less than I per
minute.
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3
and (ii) at least one wildtype tyrosine residue is replaced with an amino acid
that ~s not
capable of being phosphorylated: and comparing the level of complex formation
in the
absence of the agent to the level of complex formation in the presence of the
agent.
wherein a difference in the level of complex formation indicates the agent
alters the
interaction between the protein tyrosine phosphatase and the substrate.
In another aspect the invention provides a method of reducing the
activity of a tyrosine phosphorylated protein. comprising administering to a
subject a
substrate trapping mutant of a protein tyrosine phosphatase in which (i) the
wildtype
protein tyrosine phosphatase catalytic domain invariant aspartate residue is
replaced
with an amino acid which does not cause si~ificant alteration of the Km of the
enzyme
but which results in a reduction in Kcat to less than I per minute. and (ii)
at least one
wildtype tyrosine residue is replaced with an amino acid that is not capable
of being
phosphorylated. whereby interaction of the substrate trapping mutant protein
tyrosine
phosphatase with the tyrosine phosphorylated protein reduces the activity of
the
tyrosine phosphorylated protein. In certain embodiments the tyrosine
phosphorylated
protein is VCP: p130°S, the EGF receptor. p210 bcr:abl. VtAP kinase.
Shc (Tiganis et
al.. 1998 Llol. Cell. Biol. 18:1622-I6~-~) or the insulin receptor. In certain
other
embodiments the protein tyrosine phosphatase is PTP1B, PTP-PEST, PTPI, VLKP-1.
DEP-1. PTPu. PTPX1, PTP'Y10. SHP'_'. PTP-PEZ. PTP-VIEG1, LC-PTP, TC-PTP.
CD~~, LAR or PTPHl.
In still another aspect the invention provides a method of reducing a
transtomzing effect of at least one oncoQene associated with p130'~
phosphorylation
comprising administering to a mammal capable of expressing p130'u a substrate
trapping mutant of PTP-PEST in which (i) the wildtvpe protein tyrosine
phosphatase
catalytic domain invariant aspartate residue is replaced with an amino acid
which does
not cause significant alteration of the Km of the enzyme but which results in
a reduction
in Kcat to less than I per minute. and (ii1 at least one wildrype tyrosine
residue is
replaced with an amino acid that is not capable of being phosphorylated:
whereby the
substrate trapping mutant interacts with p130"~ to reduce the transforming
effect of at
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9
least one oncogene associated with p130"S phosphorylation. In certain
embodiments
the oncogene is v-cric, v-src or c-Ha ras.
Turning to another aspect, the present invention provides a method of
reducing formation of signaling complexes associated with p 130°x,
comprising
administering to a mammal capable of e:cpressing pI~O"~ a substrate trapping
mutant of
PTP-PEST in which (l) the wildtype protein tyrosine phosphatase catalytic
domain
invariant aspartate residue is replaced with an amino acid which does not
cause
significant alteration of the Km of the enzyme but which results in a
reduction in Kcat
to less than 1 per minute. and (ii) at least one wildtype tyrosine residue is
replaced with
an amino acid that is not capable of being phosphorylated: whereby the
substrate
trapping mutant interacts with p130'~ to reduce the formation of signaling
comple:ces
associated with p130'~.
The present invention provides. in another aspect. a method of reducing
cytotoxic effects associated with protein tyrosine phosphatase administration
or
overeYpression_ comprising administering to a mammal a substrate trapping
mutant of a
protein tyrosine phosphatase in which (l) the wildtvpe protein tyrosine
phosphatase
catalytic domain invariant aspartate residue is replaced with an amino acid
which does
not cause significant alteration of the Km of the enzyme but which results in
a reduction
in Kcat to less than 1 per minute. and (ii) at least one wildtype tyrosine
residue is
replaced with an amino acid that is not capable of being phosphorylated.
'Earning now to another aspect of the invention. there is provided an
isolated nucleic acid molecule encoding a substrate trapping mutant protein
tyrosine
phosphatase in which the wildtype protein tyrosine phosphatase catalytic
domain
invariant aspartate residue is replaced with an amino acid which does not
cause
si~ificant alteration of the Km of the enzyme but which results in a reduction
in Kcat
to less than 1 per minute: and in which at least one wildtype tyrosine residue
is replaced
with an amino acid that is not capable of being phosphoryiated. In certain
embodiments
the invention provides an antisense oligonucieotide comprising at Least 1 ~
consecutive
nucleotides complementary to the nucleic acid molecule encoding a substrate
trapping
.i0 mutant protein tyrosine phosphatase. as just described.
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It is another aspect of the invention to provide a fusion protein
comprising a poiypeptide sequence fused to a substrate trapping mutant protein
tyrosine
phosphatase in which the wildtype protein tyrosine phosphatase catalytic
domain
invariant aspartate residue is replaced with an amino acid which does not
cause
~ significant alteration of the Km of the enzyme but which results in a
reduction in Kcat
to less than 1 per minute: and in which at least one wildtype protein tyrosine
phosphazase tyrosine residue is replaced with an amino acid that is not
capable of being
phosphorylated. In certain embodiments the polypeptide is an enzyme or a
variant or
fragment thereof. In some embodiments the polypeptide sequence fused to a
substrate
10 trapping mutant protein tyrosine phosphatase is cieavable by a protease. In
certain other
embodiments the polypeptide sequence is an affinity tag polypeptide having
affinity for
a lisand.
In still another aspect. the Present invention provides a recombinant
expression construct comprising at least one promoter operably linked to a
nucleic acid
1 ~ encoding a substrate trapping mutant protein tyrosine phosphatase in which
wildtype
protein tyrosine phosphatase catalytic domain invariant aspartate residue is
replaced
with an amino acid which does not cause significant alteration of the Km of
the enzyme
but which results in a reduction in Kcat to less than I per minute: and in
which at least
one wildtype tyrosine residue is replaced with an amino acid that is not
capable of being
phosphorylated. In certain embodiments the promoter is a regulated promoter.
and in
certain other embodiments the substrate trapping mutant protein tyrosine
phosphatase is
expressed as a fusion protein with a polypeptide product of a second nucleic
acid
sequence. In certain further embodiments the polypeptide product of the second
nucleic
acid sequence is an enzyme- In certain other embodiments the expression
construct is a
5 recombinant viral expression construct In certain other embodiments the
present
invention provides a host cell comprising a recombinant expression construct
according
to those just described. In certain embodiments the host cell is a prokaryotic
cell and in
certain zmbodiments the host cell is a eukaryotic cell.
The vresent invention provides. in another aspect. a method of producing
~0 a recombinant substrate trapping mutant protein tyrosine phosphatase.
comprisin°_-
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culturing a host cell comprising a recombinant e:cpression construct
comprising at least
one promoter operably linked to a nucleic acid sequence encoding a substrate
trapping
mutant protein tyrosine phosphatase in which the wildtype protein tyrosine
phosphatase
catalytic domain invariant aspartate residue is replaced with an amino acid
which does
? not cause significant alteration of the Km of the enzyme but which results
in a reduction
in Kcat to less than 1 per minute; and in which at least one wildtype protein
tyrosine
phosphatase tyrosine residue is replaced with an amino acid that is not
capable of being
phosphoryiated. In ceztmn embodiments the promoter is a regulated promoter. In
certain other embodiments the invention provides a method of producing a
recombinant
substrate trapping mutant protein tyrosine phosphatase, comprising culturing a
host cell
infected with the recombinant viral e:cpression construct described above.
The present invention. in another aspect provides a pharmaceutical
composition comprising a substrate trapping mutant protein tyrosine
phosphatase in
which the wildtype protein tyrosine phosphatase catalytic domain invariant
aspartate
1 ~ residue is replaced with an amino acid which does not cause significant
alteration of the
Km of the enzyme but which results in a reduction in Kcat to less than 1 per
minute;
and in which at least one wildtype tyrosine residue is replaced with an amino
acid that is
not capable of being phosphorylated, in combination with a pharmaceutically
acceptable carrier or diluent.
In yet another aspect the invention provides a pharmaceutical
composition comprising an agent that interacts with a substrate trapping
mutant protein
tyrosine phosphatase in which the wildtype protein tyrosine phosphatase
catalytic
domain invariant aspartate residue is replaced with an amino acid which does
not cause
significant alteration of the Km of the enzyme but which results in a
reduction in Kcat
~5 to less than 1 per minute: and in which at least one wildtype tyrosine
residue is replaced
with an amino acid that is not capable of being phosphoryiated. in combination
with a
pharmaceutically acceptable carrier or diluent. In certain other embodiments
the
invention provides a kit for identifying a tyrosine phosphorylated protein
substrate of a
protein tyrosine phosphatase comprisin= at least one substrate trapping mutant
protein
30 tyrosine phosphatase in which ~i1 the wildtype protein tyrosine phosphatase
catalytic
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1?
domain invariant aspartate residue is replaced tenth ~ ~o mid which does not
cause
sisnincant alteration of the Km of the ezrzvrne 'nut which results in a
reduction in Kcat
to less than I pe: minute, and (ii) at least one wildtype tyrosine residue is
replaced with
an amino acid that is not capable of being phosphoryiated; and ancillary
reagents
~ suitable for use in detecting the presence or absence of a complex between
the protein
tyrosine phosphatase and a tyrosine phosphorylated protein.
These and other aspects of the present invention will become apparent
upon reference to the following deed d~~Ption and attached drawings. All
references (including websites) disclosed herein are hereby incorporated by
reference in
their entireties as if each was incorporated individually.
BRIEF DESCRIPTION OF THE DR.~WII'TGS
Figures lA-lE show a multiple amino acid sepuence alignment of the
paralytic domains of various PTPs_ the positions of amino acid residues of
PTP1B that
interact with substrate are indicated wtth small ~'owh~, and the residue
numbering
1~ at the bottom of the alignment corresponds to that for PTP1B. Figs. lA-lE
show a
taultiple sequence alignment of the catalytic domains of PTPs (SEQ ID NOS:1-
35).
Cvtosoiic eukarvotic PTPs and domain 1 of RPTPs are combined into one soup:
domains 2 of R.PTPs are in a second group and the Yersinia PTP is in a third.
Invanant
residues shared among all three gzoups are shown in lower case. Invariant and
highly
conserved residues within a group are shown is italics and bold, respectively.
Within
the Yersinia PTP sequence, residues that are either invariant or highly
conserved
between the cvtosolic and RPTP domain sc~ ~ m i~~ ~d bold., respectively.
Figure ? shows the Vmax. Kcat and Km of various PTPIB mutants
toward RCVLL (reduc=d and carboayamidomethvlated and maleylated lysozyme).
Figure ~ presents phase contrast micrographs that show
inhioition of stable NIH.",~T~ cell lines overe:cpressing PTPH1 (-. induced:
uninduc~d~.
Figure ~ presents gzowZh ctmres (mean values from triplicate plating)
that show YTowzh inhibition of stable ~~ cell lines overe~pressing PTPHI .
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l~
Figure ~ shows inhibition of cell cycle progression by PTPH1
overexpression at indicated time a$er release from hydroxyurea block. by
irnmunoblot
analysis using antibodies specific for HA epitope tag (PTPH1) or cyclin (~.
induced; -,
uninduced).
Figure 6 shows identification of pp97/VCP as a PTPH1 substrate ~n vitro
by anti-phosphotyrosine immunoblot analysis of 293 cell lysate proteins
trapped by
substrate trapping mutant PTPHl(D811A).
Figure 7 shows the amino acid sequence of pp97/V CP (nebi database
accession number Z140~) [SEQ ~ NO='~2].
Figure 8 shows identification of pp97/VCP as a PTPH1 substrate in vivo
by immunoblot analysis of ?93 cellular proteins trapped by and co-
immunoprecipitated
with substrate trapping mutant PTPH1(Y676FID811 ~).
Figure 9 shows localization of VCP tyrosine residues reco~ized by
PTPH1 to the C-terminal region of VCP.
1~ Figure 10 shows dephosphorylation of VCP in stable NIH3T3 cell lines
expressing wildtype PTPH 1.
Figure 11 shows overall profile of tyrosine phosphorylated proteins in
stable ~T3 cell lines e:cpressing wildtype PTPH1.
DEVILED DESCRIPTION OF Tl~ INV~ON
The present invention is directed to novel substrate trapping mutant
protein tyrosine phosphatases (PTPs) derived from a PTP that has been mutated
such
that the PTP catalytic domain invariant aspartate residue is replaced with an
amino acid
which does not cause si~ificant alteration of the Vtichaelis-Vlenten constant
(Km) of
the enzyme but which results in a reduction of the catalytic rate constant
(Kcat), and
25 that has further been mutated by replacement of at least one tyrosine
residue with an
amino acid that is not capable of being phosphorylated. The invention is
based. in part.
on the unexpected tinciing that under certain conditions n vivo. a PTP enzyme
may
itself undergo tyrosine phosphorylation in a manner that can alter
interactions between
the PTP and other molecules. including PTP substrates. As defined herein. a
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14
phosphatase is a member of the PTP family if it contains the si~ature motif
[I/V]HCX.aG~[S/T]G (SEQ ID X0:36). Dual specificity PTPs, i.e., PTPs which
dephosphoryiate both phosphorylated tyrosine and phosphorylated serine or
threonine,
are also suitable for use in the invention. Appropriate PTPs include. but are
not limited
to, PTP1B, PTP-PEST, PTP~I, VIKP-1, DEP-1, PTPu_ PTPY1, PTPY10, SHP?, PTP
PEZ_ PTP-~G1, LC-PTP, TC-PTP, CD~~, L~1R and PTPH1.
As noted above, substrate trapping mutant PTPs are derived from
wildtype PTPs that have been mutated such that the wildtype protein tyrosine
phosphatase catalytic domain invariant aspartate residue is replaced with an
amino acid
which does not cause significant alteration of the Km of the enzyme but which
results in
a reduction in Kcat to less than 1 per minute; and at least one wildtype
tyrosine residue
is replaced with an amino acid that is not capable of being phosphorylated. In
this
regard. amino acid sequence analysis of known PTPs reveals the presence of
twenty
seven invariant residues within the PTP pt'imaN structure (Barford et al.,
1994 Science
1~ 263:1397-1404; Jia et al., 199 Science ?68:174-178), including an aspartate
residue
in the catalytic domain that is invariant among PTP family members. When the
amino
acid sequences of multiple PTP family members are ali~ed (see, for instance.
Figure
lA-E; see also, e.g.. Barford et al., 1995 Varure Struct. Biol. 2:1043), this
invariant
aspartate residue may be readily identified in the catalytic domain region of
each PTP
sequence at a corresponding position relative to the PTP signature sequence
motif
[UV]HC~G~YR~S/T]G (SEQ ID NO:36), which is invariant among all PTPs (see.
e.o . W098/0471'_'; Flint et al.. 1997 Proc. Vat. .cad Sci. 94:1680 and
references cited
therein). However. the e.~cact amino acid sequence position numbers of
catalytic domain
invariant aspartate residues may be different from one PTP to another, due to
sequence
?5 shifts that may be imposed to ma.Yimize aliment of the various PTP
sequences (see.
e.o . Barford et al.. 199 Nature Stn~ct. Biol. ?:1043 for an alignment of
various PTP
sequenced.
In particular. portions of two PTP polypeptide sequences are regarded as
"correspondina~~ amino acid sequences_ regions. fra_ments or the like. based
on a
convention of numbering one PTP sequence according to amino acid position
number.
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
1~
and then aligning the sequence to be compared in a manner that ma.~cimizes the
number
of amino acids that match or that are conserved residues. for e:cample. that
remain polar
(e.g.. D. E, K. R. H. S. T. N, Q), hydrophobic (e.o , ~. P, V. L. I. Vt, F. W.
'~ or neutral
(e.~.. C. G) residues at each position. Similarly, a DNA sequence encoding a
candidate
PTP that is to be mutated as provided herein. or a portion. region, fragment
or the like.
may correspond to a known wildtype PTP-encoding DNA sequence according to a
convention for numbering nucleic acid sequence positions in the known wildtype
PTP
DNA sequence, whereby the candidate PTP DNA sequence is aligned with the known
PTP DNA such that at least 70%, preferably at least 80% and more preferably at
least
90% of the nucleotides in a given sequence of at least 20 consecutive
nucleotides of a
sequence are identical. In certain preferred embodiments. a candidate PTP DNA
sequence is heater than 95°'o identical to a corresponding known PTP
DNA sequence.
In certain particularly preferred embodiments, a portion, region or fragment
of a
candidate PTP DNA sequence is identical to a corresponding known PTP DNA
1 ~ sequence. As is well known in the art. an individual whose DNA contains no
irregularities (e.o , a common or prevalent form) in a particular gene
responsible for a
given trait may be said to possess a wildtype genetic complement (genotype)
for that
gene, while the presence of irregularities known as mutations in the DNA for
the gene.
for example. substitutions, insertions or deletions of one or more
nucleotides. indicates
a mutated or mutant genotype.
As noted above, in certain embodiments of the present invention there is
provided a substrate trapping mutant PTP in which catalytic domain invariant
aspartate
and at least one tyrosine residue are replaced. as provided herein.
Identification of the
catalytic domain invariant aspartate residue in PTP sequences other than those
disclosed
in Barford et al. ( 1990 may be achieved by comparing sequences using computer
alsorithms well known to those having ordinary skill in the art. such as
GENEWORKS.
Alit or the BLAST algorithm (.-~ltschul. J. :Llol. Biol. '19:~~~-~6~. 1991:
Henikotf
and Henikot~ Pror. W t1. :cad Sri. L.S~ a9:1091~-10919. 199'?. which is
available at
the ~iCBI website (http::/mywincbi.nlm.niiz.govicgi-bin/BL.~ST).
WO 00/75339 16 PCT/US00/14211
Certain embodiments of the invention pertain in part to novel PTPs in
which the invariant aspartate residue is replaced with an amino acid which
does not
cause sisnificant alteration of the Km of the enzyme but which results in a
reduction in
Kcat to less than 1 per minute (less than 1 min'). These PTPs retain the
ability to form
a complex with. or bind to. their tyrosine phosphorylated substrates. but are
catalytically
attenuated (l. e.. a substrate trapping mutant PTP retains a similar Km to
that of the
corresponding wildrype PTP, but has a Vmax which is reduced by a factor of at
least
10Z-10' relative to the wildtype enzyme, depending on the activity of the
wildtype
enzyme relative to a Kcat of less than 1 mini'). This attenuation includes
catalytic
activity which is either reduced or abolished relative to the wildtype PTP.
For example,
the invariant aspartate residue can be changed or mutated to an alanine.
valine. leucine,
isoleucine: proline. phenylalanine. tryptophan. methionine. glycine. serine.
threonine,
cysteine, tyrosine. aspan~;n_e, ~utamine. lysine, ar~nine or histidine.
The preferred substrate trapping mutant PTPs described herein, in which
1 ~ the invariant aspartate residue is replaced with an amino acid which does
not cause
significant alteration of the Km of the enzyme but which results in a
reduction in Kcat
to less than 1 per minute (less than 1 min'), and in which at least one
tyrosine residue is
replaced with an amino acid that is not capable of being phosphorylated, may
further
comprise other mutations. In particularly preferred embodiments. such
additional
mutations relate to substitutions. insertions or deletions (most preferably
substitutions)
that assist in stabilizing the PT'Plsubstrate complex. For example, mutation
of the
serine,%threonine residue in the si~ati~re motif to an alanine residue (S/T~ ~
mutant)
may change the rate-determining step of the PTP-mediated substrate
dephosphorylation
reaction. For the unmodified PTP, formation of the transition state may be
rate-
?5 limiting, whereas in the case of the S~T~:~ mutant the breakdown of the
transition
state may become rate-Limiting, thereby stabilizing the PTP/substrate complex.
Such
mutations may be valuably combined with the replacement of the PTP catalytic
domain
invariant aspartate residue and the replacement of PTP tyrosine as provided
herein. for
example_ with re?ard to stabilizing the PTP-substrate complex and facilitating
its
.0 isolation. :~s another e:cample. substitution of any one or more other
amino acids
CA 02375145 2001-11-23
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
17
present in the wildtype PTP that are capable of being phosphoryiated as
provided herein
(e.g.. serine. threonine, tyrosine) with an amino acid that is not capable of
being
phosphorylated may be desirable, with regard to the stability of a PTP-
substrate
complex.
As noted above, the present invention provides substrate trapping mutant
PTPs in which catalytic domain invariant aspartate and at least one tyrosine
residue are
replaced, wherein the tyrosine is replaced with an amino acid that is not
capable of
being phosphorylated. The amino acid that is not capable of being
phosphorylated may.
in prefezred embodiments, be alanine, cysteine, aspartic acid, glutamine,
glutamic acid,
phenylalanine, glycine, histidine, isoleucine, lysine. leucine, methionine,
asparagine,
proline. ar~inine. valise or trvptophan. The desirability of the tyrosine
replacement
derives from the surprising observation that under certain conditions in vivo.
a PTP
enzyme may itself underjo tyrosine phosphorylation in a manner that can alter
interactions between the PTP and other molecules. including PTP substrates.
PTP
substrates include any naturally or non-naturally tyrosine-phosphorylated
peptide,
polypeptide or protein that can specifically bind to and/or be
dephosphorylated by a
PTP as provided herein. Thus, replacement of a tyrosine residue found in the
wildtype
amino acid sequence of a particular PTP with another amino acid as provided
herein
stabilizes a complex formed'ov the subject invention substrate trapping mutant
P'?'P and
a PTP substrate when the amount of complex that is present and/or the affinity
of the
mutant PTP for the substrate increases_ relative to comple:c formation using a
PTP in
which the tyrosine residue is not replaced
As noted above, the present invention exploits the substrate trapping
mutant PTPs described herein to provide a method of identifying a tyrosine
?5 phosphorylated protein that is a substrate of a wildtype PTP. According to
this aspect
of the invention_ a sample comprisins at least one tyrosine phosphorylated
protein is
combined with at least one substrate trapping mutant PTP as provided herein.
and the
presence or absence of a complex comprising the substrate and the mutant PTP
is
determined. T'ne binding inte:action between a PTP and a PTP substrate may
result in
;0 the formation of a complex. which refers to the affinity interaction of the
PTP and the
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
is
PTP substrate. A complex may include a sigria,ling complex. which refers to
any
complex that by virtue of its formation. its stable association and/or its
dissociation
directly or indirectly provides a biological signal. Such signals may include.
for
example by way of illustration and not limitation. intracellular and/or
intercellular
events that lead to molecular binding. covalent or non-covalent modification
of
molecular structure, gene expression. genetic recombination. ?enetic
integration,
nucleic acid synthesis or subcellular particle assembly. and may also include
endocvtic.
phagocvtic. nucleolytic, proteolvtic. lipolvtic. hydrolytic. catalytic. or
other regulatory
events.
Determination of the presence of a stable complex between a PTP and a
PTP substrate refers to the use of any methodology known in the art for
demonstrating
an intermolecular interaction between a PTP and a PTP substrate according to
the
present disclosure. Such methodologies may include. by way of illustration and
not
limitation. co-purification, co-precipitation, co-immunoprecipitation,
radiometric or
16 ffuorimetric assays, western immunoblot analyses. amity capture including
atf~ty
techniques such as solid-phase ligand-counteriigand sorbent techniques,
affinity'
chromato~aphy and surface affinity plasmon resonance, and the like. For these
and
other useful affnity techniques, see. for example. Scopes. R.K., Protein
Purification:
Principles and Practice. 1987. Springer-Veriag. NY: Weir. D.M., Handbook of
Erperimental Immunology, 1986, Blackwell Scientific. Boston; and Hermanson.
G.T. et
al.. Immobili=ed .-lffrniry Ligand Techniques, 1992. Academic Press, Inc.,
California:
which are hereby incorporated by reference in their entireties, for details
regarding
techniques for isolating and chatacteriang complexes. including affinity
techniques. A
PTP may interact with a PTP substrate via specific binding if the PTP binds
the
substrate with a Ka of heater than or eq~ to bout 10'~ M-l. preferably of
'eater than
or equal to about 10= 1~L-l, more preferably of greater than or equal to about
106 Vl-I
and still more preferably of Greater than or equal to about 10 % VI-~ to 10~ M-
' . .Wfinities
of binding partners such as a PTP and a PTl' substrate can be readily
determined using
conventional techniques. for ~:campie those desc:ibed by Scatchard et al.. .-
inn. V. Y.
:0 .cad Sci. ~ 1:660 ~19~9).
CA 02375145 2001-11-23
WO 00/75339 19 PCT/US00/14211
Without wishing to be bound by theory, it is contemplated that
phosphorylated tyrosine residues that are part of a PTP molecule itself may
influence
the interaction between the PTP molecule and PTP substrate molecules. which
include
tyrosine phosphorylated proteins that a PTP may bind and/or dephosphorylate.
:according to this non-limiting theory. a conserved tyrosine residue present
in a PTP
primary structure may be a receptor for transfer of a phosphate soup from the
highly
reactive thiophosphaze intermediate that may be formed between the invariant
cysteine
residue found in the simature motif that resides in the active site of the PTP
catalytic
domain (as described above) and the phosphate soup present in the form of
phosphotyrosine on the PTP substrate phosphoproteirL Thus. although a
conserved
tyrosine residue in a PTP active site may facilitate intermolecular
orientation of the PTP
relative to its substrate by providing a hydrophobic interaction with the
substrate
phosphotyrosine, and may further act as a phosphate acceptor. the invention is
not so
Limited.
1 ~ As described above. the present invention provides a mutated PTP in
which at least one tyrosine residue is replaced with an amino acid that cannot
be
phosphorylated. Preferably the tyrosine residue is located in the PTP
catalytic domain,
which refers to the approximately 250 amino acid region that is highly
conserved
among the various PTPs. as noted above (see also, e.o., Barford_ 1998 ~rrn.
Rev.
~0 Biophys. Biomol. Struct. ~7:1~3; Jia_ 1997 Biochem. Cell Biol. 75:17; Van
Vactor et al..
1998 Curr. Opin Genet. bevel. 8:112) Vlore preferably. the tyrosine residue is
located
in a PTP active site. which refers to the region within the PTP catalytic
domain that
contains the PTP si~ature motif and which also includes those amino acids that
form
the PTP binding site pocket or "cradle' for substrate binding and
dephosphorylation.
25 further inciudinQ the invariant aspastate-containing loop (when present)
and adjacent
peptide backbone sequences that contribute to substrate reco~ition and
catalysis (see.
e.g.. Jia_ 1997. In a most preferred embodiment. the tyrosine residue is
replaced with
phenvlalanine. and in another most preferred embodiment. the tyrosine residue
is a
conserved residue that corresponds to the tyrosine situated at position 676 in
the amino
;0 acid sequence of human PTPH1. and which also corresponds to the amino acid
residue
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
at position ~6 in the PTP-1B sequence shown in Figure 1. In other preferred
embodiments, the tyrosine residue is a PTP conserved residue. which includes
tyrosine
residues that are present at corresponding positions within two or more PTP
amino acid
sequences relative to the position of the signature motif. In other preferred
5 embodiments. the tyrosine residue is replaced with an amino acid that
stabilizes a
comple:c formed by the PTP and at least one substrate molecule. as provided
herein.
~s noted above. PTPs that may be useful according to the present
invention include any PTP which has an invariant aspartate residue in a
corresponding
position in the catalytic domain. and a tyrosine residue. By way of
illustration and not
10 limitation, in certain preferred embodiments of the present invention, the
substrate
trapping mutant PTP has at least one tyrosine residue found in the
corresponding
wildtype sequence replaced with phenwlalanine. In certain particularly
preferred
embodiments. the PTP is PTPH1 having the invariant aspartate replaced by
alanine and
the tyrosine at position 676 replaced by phenylalanine. PTPH1(Y676F/D811A). In
1 ~ certain other embodiments. the PTP is a mutated PTP-PEST phosphatase in
which the
cysteine found in the corresponding wildtype sequence is replaced with serine
and at
Least one wildtype tyrosine residue is replaced with an amino acid that cannot
be
phosphorylated. It should be recognized. however. that mutant PTPs other than
those
specifically desr:ibed herein can readily be made by ali~ing the amino acid
sequence
?0 of a PTP catalytic domain with the amino acid sequence of PTPs that are
described
herein (including those provided by the cited references), identifying the
catalytic
domain invariant aspartate residue and at least one tyrosine residue. and
changing these
residues. for e:cample by site-directed mutagenesis of DNA encoding the PTP.
Vfodification of DNA may be performed by a variety of methods.
'_'S inciudin= site-specinc or site-,iirected mutagenesis of DNA sncodina the
PTP and the
use of DNA amplification methods using primers to introduce and amplify
alterations in
the DN -~ template. such as PCR splicing by overlap extension (SOE). Site-
directed
mutagenesis is t~~picallv effected using a phaQe vector that has single- and
doubie
stranded forms. such as Vt 13 phage vectors. which are well-~now-n and
commercially
~0 available. Other suitable vectors that contain a single-stranded phaae
origin of
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
21
replication may be used (see. e.o . Veira et al.. .Meth. En..-ymol. 1 ~ :3.
1987). In General,
site-directed mutagenesis is performed by preparing a single-stranded vector
that
encodes the protein of interest (e.o . a member of the PTP family). :fin
oligonucieotide
primer that contains the desired mutation within a re°_-ion of homology
to the DNA in
the single-stranded vector is annealed to the vector followed by addition of a
DNA
polymetase, such as E toll DNA polymerase I (Klenow fragment), which uses the
double stranded region as a primer to produce a hetezoduplex in which one
strand
encodes the altered sequence and the other the original sequence. Additional
disclosure
relating to site-directed mutagenesis may be found, for example, in Kunkel et
GI.
10. (ll~Ierhods in Er~ymol. 1 ~~:367, 1987); and in U.S. Patent Nos. ~.~ 18.84
and
4,737,462. The heteroduplex is introduced into appropriate bacterial cells.
and clones
that include the desired mutation are selected. The resulting altered DNA
molecules
may be expressed recombinantly in appropriate host cells to produce the
modified
protein.
1~ Specific substitutions of individual amino acids through introduction of
site-directed mutations are well-known and may be made according to
methodologies
with which those having ordinary skill in the art will be familiar. The
effects on
catalytic activity of the resulting mutant PTP may be determined empirically
merely by
testing the resulting modified protein for the preservation of the Km and
reduction of
20 Kcat to less than 1 per minute as provided herein and as previously
disclosed (e.g.,
W098l04712; Flint et al.. 1997 Proc. iVat. .cad Sci. 94:1680). The effects on
the
ability to tyrosine phosphorylate the resulting mutant PTP molecule can also
be
determined empirically merely by testing such a mutant for the presence of
phosphotyrosine. as also provided herein. for example. following exposure of
the
mutant to conditions in vitro or in vivo where it may acs as a PTK acceptor.
Although the specific examples of PTP mutants described below are DA
(aspartate to alanine) mutants. YF (tyrosine to phenylalanine) mutants. CS
mutants and
combinations thereof, it will be understood that the subject invention
substrate trapping
mutant PTPs are not limited to these amino acid substitutions. The invariant
aspartate
30 residue can be changed. for example by site-directed mutaaenesis. to anv
amino acid
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
that does not cause si~ificant alteration of the Kl-n of the enzyme but which
results in a
reduction in Kcat to less than 1 per minute (less than 1 min'). For example.
the
invariant aspartate residue can be changed or mutated to an alanine. valine_
leucine_
isoleucine: proline. phenylalanine, tryptophan, methionine, glycine. serine.
threonine,
S cysteine. tyrosine. asparagine. ~lutamine_ lysine. ar~nine or histidine, or
other natural
or non-natural amino acids known in the art inciudina derivatives. variants
and the like.
Similarly. substitution of at least one tyrosine residue may be with any amino
acid that
is not capable of being phosphorylated (i.e.. stable. covalent modification of
an amino
acid side chain at a hydroxyl with a phosP~e pup), for example alanine,
cysteine_
aspartic acid. giutamine, gluutamic acid, phenyialanine. ~ycine, histidine,
isoleucine,
lysine, leucine_ methionine. asparagine, proline, arQinine, valine or
tryptophan_ or other
natural or non-natural amino acids known in the art inciudiny derivatives.
variants and
the like.
The nucleic acids of the present invention may be in the form of R.hIA or
in the form of DNA. which DNA includes cDNA. aenomic DNA, and synthetic DNA.
The DNA may be double-stranded or single-stranded. and if single stranded may
be the
coding strand or non-coding (anti-sense) strand. A nucleic acid molecule
encoding a
substrate trapping mutant PTP in which the wildtype protein tyrosine
phosphatase
catalytic domain invariant aspartate residue is replaced with an amino acid
which does
?0 not cause si~ificant alteration of the Km of the enzyme but which results
in a reduction
in Kcat to less than 1 per minute. and in which at least one wildtype tyrosine
residue is
replaced with an amino acid that is not capable of being phosphorylated. may
be
identical to the coding sequence known in the art for any even PTP. as
described
above. or may be a different coding sequence. which. as a result of the
redundancy or
de~eneracv of the genetic code. encodes the same PTP.
The present invention fiirther relates to variants of the herein described
nucleic acids which encode fragznents_ analogs and derivatives of a PTP
polypeptide_
includinz a mutated PTP such as a substrate trappins mutant PTP. T'ne variants
of the
nucleic acids eacodins PTPs may be naturally occuring allelic variants of the
nucleic
;0 acids or non-naturally occurring variants. As is known in the art. an
allelic variant is an
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
alternate form of a nucleic acid sequence which may have at least one of a
substitution,
a deletion or an addition of one or more nucleotides. any of which does not
substantially
alter the function of the encoded PTP polvpeptide.
Equivalent DNA constructs that encode various additions or
substitutions of amino acid residues or sequences. or deletions of terminal or
internal
residues or sequences not needed for biological activity are also encompassed
by the
invention. For example, sequences encoding Cys residues that are not essential
for
biological activity can be altered to cause the Cys residues to be deleted or
replaced
with other amino acids. preventing formation of incorrect intramolecular
disulfide
bridges upon renaturation. Other equivalents can be prepared by modification
of
adjacent dibasic amino acid residues to enhance expression in yeast systems in
which
KE:~ protease activity is present. EP ~ 1?.91-~ discloses the use of site-
specific
mutagenesis to inactivate KEG prot~e processing sites in a protein. KEG
protease
processing sites are inactivated by deleting, adding or substituting residues
to alter :~rg-
Arg, Arg-Lys, and Lys-__.~rg pairs to eliminate the occurrence of these
adjacent basic
residues. Lys-Lys pairings are considerably less susceptible to KEY' cleavage.
and
conversion of erg-Lys or Lys-erg to Lvs-Lys represents a conservative and
preferred
approach to inactivatin°_- KE.~? sites.
The present invention further relates to PTP polypeptides including
substrate trapping mutant PTPs. and in particular to methods for producing
recombinant
pTP polypeptides by culturing host cells containing PTP expression constructs.
and to
isolated recombinant PTP polypeptides. The polvpeptides and nucleic acids of
the
present invention are preferably provided is an isolated form. and in certain
preferred
embodiments are purified to homogeneity. The terms "fra~nent.~ ''derivative''
and
-=~~og" when referring to PTP poiypeptides or fusion proteins. including
substrate
trapping mutant PTPs. refers to any PTP polypeptide or fusion protein that
retains
essentially the same biological function or activity as such polypeptide. Thus-
an
analog includes a proprotein which c :n be activated by cleavage of the
proprotein
portion to produce an active PTP polypeptide. The polypeptides of the present
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
?4
invention may be recombinant polvpeptides or synthetic polypeptides. and are
preferably recombinant polvpeptides.
;~ fira~ent derivative or analog of a PTP polvpeptide or fusion protein.
including substrate trapping mutant PTPs. may be (l) one in which one or more
of the
amino acid residues are substituted with a conserved or non-conserved amino
acid
residue (preferably a conserved amino acid residue), and such substituted
amino acid
residue may or may not be one encoded by the genetic code. or (ii) one in
which one or
more of the amino acid residues includes a substituent soup. or (iii) one in
which the
PTP polypeptide is fused with another compound. such as a compound to increase
the
half life of the polvpeptide (e.o , polyethylene glycol), or (iv) one in which
additional
amino acids are fused to the PTP polypeptide. including amino acids that are
employed
for purification of the PTP polypeptide or a proprotein sequence. Such
fragments.
derivatives and analogs are deemed to be within the scope of those skilled in
the art
from the teachings herein.
1~ The polypeptides of the present invention include PTP polypeptides and
fusion proteins having amino acid sequences that are identical or similar to
PTP
sequences known in the art. For e:cample by way of illustration and not
limitation- the
human PTP polypeptides (including substrate trapping mutant PTPs) referred to
below
in the E.~camples are contemplated for use according to the instant invention.
as are
polypeptides having at least 70% similarity (preferably 70% identity), more
preferably
90°,'° similarity (more preferably 90% identity) and still more
preferably 95% similarity
(still more preferably 9~°,% identity) to the polypeptides described in
references cited
herein and in the E-'tamPles and to portions of such polypeptides. wherein
such portions
of a PTP polypeptide generally contain at least 30 amino acids and more
preferably at
5 least ~ 0 amino acids.
As knowm in the art "similarity" between two polypeptides is determined
by comparins the amino acid sequence and conserved amino acid substitutes
thereto of
the polvpeptide -to the sequence of a second polvpeptide (e.o . using
GE~1EWORKS.
:~li~ or the BL:~ST algorithm. as described abovel. Fra~nents or portions of
the
;0 polypeptides of the present invention may be employed for producing the
corresponding
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
?5
full-length poiypeptide by peptide synthesis; therefore. the fragments may be
employed
as intermediates for producing the full-length polvpeptides. Fragments or
portions of
the nucleic acids of the present invention may be used to synthesize full-
length nucleic
acids of the present invennon.
The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
e:cample, a
naturally occurring nucleic acid or polypeptide present in a living animal is
not isolated.
but the same nucleic acid or polypeptide, separated from some or all of the co-
e:cisting
materials in the natural system, is isolated. Such nucleic acid could be part
of a vector
and/or such nucleic acid or polvpeptide could be part of a composition. and
still be
isolated in that such vector or composition is not part of the natural
environment for the
nucleic acid or polypeptide. The term "gene' means the se~nent of DNA involved
in
producing a polypeptide chain: it includes regions preceding and following the
coding
region "leader and trailer' as well as intervening sequences (introns) between
individual
1~ coding segments (e:cons).
As described herein. the invention provides a fusion protein comprising a
polvpeptide fused to a substrate trapping mutant PTP in which the wildtype
protein
tyrosine phosphatase catalytic domain invariant aspartate residue is replaced
with an
amino acid which does not cause significant alteration of the Km of the enzyme
but
?p which results in a reduction in Kcal to less than 1 pe: minute, and in
which at least one
wildtype protein tyrosine phosphatase tyrosine residue is replaced with an
amino acid
that is not capable of being phosphorylated. Such PTP fusion proteins are
encoded by
nucleic acids have the substrate trapping mutant PTP coding sequence fused in
frame to
an additional coding sequence to provide for e:cpression of a PTP polypeptide
sequence
fused to an additional functional or non-functional polvpeptide sequence that
permits.
for e:campie by way of illustration and not Limitation. detection. isolation
and/or
purification of the PTP fusion protein. Such PTP fusion proteins may permit
detection.
isolation and/or purification of the PTP fusion protein by protein-protein
ai~nity. metal
affinity or charge atFnitv-based polypeptide purincation. or by specific
protease
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
26
cleavage of a fusion protein containing a fusion sequence that is cleavable by
a protease
such that the PTP polypeptide is separable from the fusion protein.
Thus. PTP fusion proteins may comprise affinity tag polypeptide
sequences. which :efers to polypeptides or peptides added to PTP to facilitate
detection
and isolation of the PTP via a specific affinity interaction with a ligand.
The ligand
may be any molecule. receptor, counterreceptor, antibody or the like with
which the
a~niry tag may interact through a specific binding interaction as provided
herein. Such
peptides include. for e:cample, poly-His or the antigenic identification
peptides
described in U.S. Patent No. ~,011.9I? and in Hopp et al., (1988 BiolTechnolo~
6:1204), or the ~'RESST" epitope tag (Invitrogen, Carisbad, CA). The affinity
sequene~ may be a he:ca-histidine tag as supplied. for e:cample, by a pBAD~His
(Invitrogen) or a pQE-9 vector to provide for purification of the mature
polypeptide
fused to the marker in the case of a bacterial host or. for e:cample, the
af~lnity sequence
may be a hemagglutinin (HA) tag when a mammalian host e.o., COS-7 cells, is
used.
1 ~ The HA tag corresponds to an antibody defined epitope derived from the
influenza
hemagglutinin protein (Wilson et al.. 198- Cell 3 i :767).
PTP fusion proteins may further comprise immunoglobulin constant
region polypeptides added to PTP to facilitate detection, isolation andlor
localization of
PTP: The immunoglobulin constant region polvpeptide preferably is fused to the
C-
terminus of a PTP polypeptide. General preparation of fusion proteins
comprising
heterologous polvpeptides fused to various portions of antibody-derived
polypeptides
(including the Fc domain) has been described_ e.o , by Ashkenazi et al. (PV:~S
v;Srl
88: I0~35. 1991) and Byre et al. (Nature 3-~-1:677, 1990). A gene fusion
encoding the
PTP:Fc fusion protein is inserted into an appropriate e:cpression vector. In
certain
?5 embodiments of the invention. PTP:Fc fusion proteins may be allowed to
assemble
much like antibody molecules. whereupon interchain disulfide bonds form
between Fc
polvpeptides. yielding dimeric PTP fusion proteins.
. PTP fusion proteins having Specific binding affinities for pre-selected
antigens by virtue of fusion polypeptides comprising immunoglobulin V-re?ion
domains encoded by DNA sequences -linked in-frame to sequences encoding PTP
are
CA 02375145 2001-11-23
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''7
also within the scope of the invention. including variants and fragments
thereof as
provided herein. General strategies for the construction of fusion proteins
having
immunoglobulin V-region fusion polvpeptides are disclosed. for example, in EP
0318664; U.S. 6,132,406; U.S. 6,091,6 I3; and U.S. ~.-X76.786.
6 The nucleic acid of the present invention may also encode a fusion
protein comprising a PTP polypeptide fused to other polypeptides having
desirable
affinity properties. for example an enzyme such as glutathione-S-transferase.
As
another example, PTP fusion proteins may also comprise a PTP polypeptide fused
to a
Staphylococcus aurezrs protein A polypeptide; protein :~ encoding nucleic
acids and
their use in constructing fusion proteins having affinity for immunoglobulin
constant
regions are disclosed generally, for example, in U.S. Patent 5.100,788. Other
useful
affinity polypetides for construction of PTP fusion proteins may include
streptavidin
fusion proteins, as disclosed. for example. in WO 89/03422; U.S. ~,-X89>>28;
U.S.
6,672,691; WO 93/24631; U.S. 6,168,049; U.S. 6?72:~5~ and elsewhere, and
avidin
fusion proteins (see, e.j . EP 611,747). r1s provided herein and in the cited
references,
PTP polypeptide sequences. including substrate trapping mutant PTPs, may be
fused to
fusion polypeptide sequences that may be full length fusion polypeptides and
that may
alternatively be variants or fragments thereof.
The present invention also contemplates PTP fusion proteins that contain
polypeptide sequences that direct the fusion protein to the cell nucleus. to
reside in the
lumen of the endoplasmic reticulum (ER), to be secreted from a cell via the
classical
ER-Golgi secretory pathway (see. e.o . von Heijne, J. ~Llembrane Biol. 11 ~
:195-201,
1990), to be incorporated into the plasma membrane, to associate with a
specific
evtoplasmic component including the cvtoplasmic domain of a transmembrane cell
?5 surface receptor or to be directed to a particular subcellular location by
any of a variety
of known intracellular protein sorring mechanisms with which those skilled in
the art
will be familiar (See. e.o.. Rothman. .Vature X72:66-63. 1994. :~drani et al..
1998 .I.
Biol: C~rem. '_'73:10 17. and references cited therein.). Accordingly. these
and related
embodiments are ;.ncompassed by the instant compositions and methods directed
to
CA 02375145 2001-11-23
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~s
targeting a polypeptide of interest to a predefined intracellular. membrane or
extracellular localization.
The present invention also relates to vectors and to constructs that
include nucleic acids of the present invention. and in particular to
"recombinant
expression constructs' that include any nucleic acids encoding PTP
polypeptides
according to the invention as provided above; to host cells which are
genetically
engineered with vectors and/or constructs of the invention and to the
production of PTP
polypeptides and fusion proteins of the invention. or fragments or variants
thereof. by
recombinant techniques. PTP proteins can be expressed in mammalian cells.
yeast
bacteria, or other cells under the control of appropriate promoters. Cell-free
translation
systems can also be employed to produce such proteins using RIVAs derived from
the
DNA constructs of the present invention. appropriate cloning and expression
vectors
for use with prokaryotic and eukaryotic hosts are described. for example. by
Sambrook,
et al.; :Llolecular Cloning: <~ Laboratory Vlarruah Second Edition. Cold
Spring Harbor.
New York, (1989).
Generally. recombinant expression vectors will include origins of
replication and selectable markers permitting transformation of the host cell,
e.g.. the
ampicillin resistance gene of E. coli gad S cerevisiae TRPl gene, and a
promoter
derived from a highly-expressed gene to direct transcription of a downstream
structural
sequence. Such promoters can be derived from operons encoding glycolytic
enzymes
such as 3-phosphoglycerate kinase (PGK), a-factor. acid phosphatase, or heat
shock
proteins_ among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination sequences.
Optionally. the
heterologous sequence can encode a fusion protein including an N-terminal
?5 identification peptide imparting desired characteristics. e.o .
stabilization or simplified
purification of expressed recombinant product.
L: seful expression constructs for bacterial use are constructed by
inserting into an expression vector a structural DNh sequence encoding a
desired
protein toge~.her with suitable translation initiation and termination sisals
in operable
~0 reading phase with a functional promoter. The construct may comprise one or
more
CA 02375145 2001-11-23
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?9
phenotypic selectable markers and an origin of replication to ensure
maintenance of the
vector construct and. if desirable. to provide amplification within the host.
Suitable
prokaryotic hosts for transformation include E. coll. Bacillus subrilis,
Salmonella
typhimurium and various species within the genera Pseudomonas. Streptomyces.
and
Staphylococcus. although others may also be employed as a matter of choice.
:any
other plasmid or vector may be used as long as they are replicable and viable
in the
host
As a representative but nonlimiting example. useful expression vectors
for bacterial use can comprise a selectable marker and bacterial origin of
replication
derived from commercially available plasmids comprising genetic elements of
the well
known cloning vector pBR~2? (ATCC 3701'x. Such commercial vectors include. for
e,t~ple, p~?~;_; (pharmacia Fine Chemicals. Uppsala. Sweden) and GEM1
(Promega Biotec, Madison. Wisconsin. USA). These PBR:p~ ~~backbone'' sections
are
combined with an appropnate promoter and the structural sequence to be
expressed.
1 j Following transformation of a suitable host strain and growth of the host
strain to an appropriate cell density. the selected promoter, if it is a
regulated promoter
as provided herein, is induced by appropriate means (e.g.. temperature shift
or chemical
induction) and cells are cultured for an additional period. Cells are
typically hariested
by centrifugation, disrupted by physical or chemical means, and the resulting
c:ude
extract retained for further purification. Microbial cells employed in
expression of
proteins can be disrupted by any convenient method, including freeze-thaw
cycling,
sonication. mechanical disruption- or use of cell lysing agents; such methods
are well
how to those skilled in the art.
Thus. for example. the nucleic acids of the invention as provided herein
may be included in anv one of a variety of expression vector constructs as a
recombinant expression construct for expressing a PTP polypeptide. Such
vectors and
constructs include chromosomal. nonchromosomal and synthetic DNA sequences. e-
,g..
derivatives of SV -10: bacterial plasmids: phage DN.-~: baculovirus: vesst
plasmids:
vectors derived from combinations of plasmids and phage DNA.. viral DNA. such
as
;0 vaccinia. adenovirus. fowl pox virus. and pseudorabies. However. any other
vector may
CA 02375145 2001-11-23
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be used for preparation of a recombinant expression construct as long as it is
replicable
and viable in the host.
The appropriate DNA sequences) may be inserted into the vector by a
variety of procedures. In general, the DNA sequence is inserted into an
appropriate
5 restriction endonuciease sites) by procedures known in the art. Standard
techniques for
cloning, DNA isolation, amplification and purification. far enzymatic
reactions
involving DNA ligase. DNA polymezase, restriction endonucleases and the like.
and
various separation techniques are those known and commonly employed by those
skilled is the art. A number of standard techniques are described, for
example. in
IO Ausubel et al. (1993 Current Protocols in Llolecular Biology, Greene Publ.
P.ssoc. Inc.
& John Wiiey 3t Sons. Inc., Boston. ~); S~brook et al. (1989 :Ltolecular
Cloning,
Second Ed., Cold Spring Harbor Laboratory. Piainview. iV~; Maniatis et al.
(1982
Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, ~; and elsewhere.
The DNA sequence in the expression vector is operatively linked to at
l~ least one appropriate expression control sequences (e.g.. a promoter or a
regulated
promoter) to direct mRNA synthesis. Representative e.~camples of such
expression
control sequences include LTR or SV.~O promoter. the E cvli lac or trp. the
phage
lambda. P~ promoter and other promoters known to control expression of genes
in
prokaryotic or eukaryotic cells or their viruses. Promoter regions can be
selected from
20 _any desired gene using C~-~T (chloramphenicol transferase) vectors or
other vectors with
selectable markers. Two appropriate vectors are pKK? i2-8 and pCVi7.
Particular
named bacterial promoters include lack lacZ. T~, T7, apt, lambda P~, P:. ~d
nP'
Eukaryotic promoters include CVIV immediate early. HSV thymidine kinase, early
and
late SV40, LTRs from retrovirus. and mouse metallothionein-I. Selecnon of the
appropriate vector and promoter is well within the level of ordinary skill in
the art, ~d
preparation of certain particularly preferred recombinant expression
constructs
comprising at least one promoter or regulated promoter operably linked to a
nucleic
acid encoding a PTP polvpeptide is described herein.
:~s noted'above. in certain embodiments the vector may be a viral vector
;0 such as a retroviral vector. For example. retroviruses from which the
retroviral plasmid
CA 02375145 2001-11-23
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;l
vectors may be derived include. but are not limited to, Moloney Marine
Leukemia
Virus. spleen necrosis virus. retroviruses such as Rous Sarcoma Virus. Harvey
Sarcoma
virus. avian leukosis virus. gibbon ape leukemia virus. human immunodeficiency
virus.
adenovirus. Myeioproliferative Sarcoma V leas, and mammary tumor virus.
The viral vector includes one or more promoters. Suitable promoters
which may be employed include, but are not limited to, the retroviral LTR; the
SV=10
promoter, and the human cytomegalovirus (CAN) promoter described in Miller. et
al..
Biotechniques % :980-990 ( 1989), or any other promoter (e.o , cellular
promoters such as
eukaryotic cellular promoters including, but not limited to, the histone, pol
III. and (3-
actin promoters). Other viral promoters which may be employed include. but are
not
limited to, adenovitus promoters. thymidine kinase (TK) promoters. and B 19
parvovirus
promoters. The selection of a suitable promoter will be apparent to those
skilled in the
art from the teachinss contained herein. and may be from among either
regulated
promoters or promoters as described above.
1 ~ The retroviral plasmid vector is employed to transduce pac.~caging cell
lines to form producer cell lines. Examples of packaging cells which may be
transfected include, but are not limited to. the PE~O1, P.A l 17. v~-2, yr-
AUI. PAl?, T19-
1~Y, VT-19-17-H2, wCRE, y~CRIP. GP+E-86. GPlenvAml'_'. and D~~1 cell lines as
described in Miller. F~uman Gene Tnerapv, 1:~-l~ (1990), which is incorporated
herein
by reference in its entirety. The vector may transduce the packaging cells
through any
means known in the art. Such means include. but are not limited to,
eiectroporation. the
use of liposomes, and calcium phosphate precipitation. In one alternative. the
retroviral
plasmid vector may be encapsulated into a liposome. or coupled to a lipid. and
then
administered to a host.
The producer cell line Qenerates infectious retroviral vector particles
which include the nucleic acid sequences) encoding the PTP polypeptides or
fusion
proteins. Such retroviral vector particles then may be employed- to transduce
eukarvotic cells. either in vitro or in vivo. The transduced eukaryotic cells
will e~cpress
the nucleic acid sequencet s1 encoding the PTP polypeptide or fusion protein.
Eukaryotic cells which may be transduced include. but are not limited to.
embryonic
CA 02375145 2001-11-23
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stem cells, embryonic carcinoma cells. as well as hematopoietic stem cells,
hepatocytes.
fibroblasu. myoblasu, keratinocytes. endothelial cells. bronchial epithelial
cells and
various other culture-adapted cell lines.
As another example of an embodiment of the invention in which a viral
vector is used to prepare the recombinant PTP expression construct in one
preferred
embodiment. host cells transduced by a recombinant viral construct directing
the
expression of PTP polypeptides or fusion proteins may produce viral particles
containing expressed PTP polvpeptides or fusion proteins that are derived from
portions
of a host cell membrane incorporated by the viral particles during viral
budding. In
another preferred embodiment, PTP encoding nucleic acid sequences are cloned
into a
baculovirus shuttle vector, which is then recombined with a baculovirus to
generate a
recombinant baculovirus expression constrict that is used to infect. for
example. S~
host cells. as described in Baculovirus Expression Protocols. ~l~lethods in
.Llolecular
Biology Vol. 39. Christopher D. Richardson, Editor, Human Press, Totowa, NJ,
1995;
piwnica Worms, ''E.Ypression of Proteins in Insect Cells Using Baculoviral
Vectors;'
Section II in Chapter 16 in: Short Protocols in :Llolecular Biology. 2"d Ed.,
Ausubel et
al., eds.. John Wilev & Sons. New York_ New York, 199?, pages 16-32 to 16-~8.
In another aspect the present invention relates to host cells containing
the above described recombinant PTP expression consmicu. Host cells are
genetically
?0 engineered (transduced, transformed or transfected) with the vectors and/or
expression
constructs of this invention which may be. for example, a cloning vector. a
shuttle
vector or an expression construct The vector or construct may be, for
e:cample. in the
form of a plasmid. a viral particle. a phage. etc. The engineered host cells
can be
cultured in conventional nutrient media modified as appropriate for activating
~5 promoters. selecting transformanu or amplifying particular genes such as
genes
encoding PTP polypeptides or PTP fusion proteins. The culture conditions for
particular host cells selected for zxpression. such as temperature. pH and the
like. will
be readily apparent ~o the ordinarily skilled artisan.
The host cell can be a higher eukaryotic cell. such as a mammalian ce! 1.
~0 or a lower eukar<<otic cell. such as a yeast cell. or the host cell can be
a prokaryotic cell.
CA 02375145 2001-11-23
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>;
such as a bacterial cell. Representative examples of appropriate host cells
according to
the present invention include, but need not be limited to, bacterial cells,
such as E. coli.
Streptomyces. Salmonella typhimurium; fungal cells. such as yeast: insect
cells. such as
Drosophila S2 and Spodoptera SfP; animal cells. such as CHO> COS or ?9S cells;
adenoviruses; plant cells. or any suitable cell already adapted to in vitro
propagadon or
so established de novo. The selection of an appropriate host is deemed to be
within the
scope of those skilled in the art from the teachings herein.
Various mammalian cell culture systems can also be employed to
express recombinant protein. The invention is therefore directed in part to a
method of
producing a recombinant substrate trapping mutant protein tyrosine
phosphatase. by
culturing a host cell comprising a recombinant expression construct that
comprises at
least one promote: operably linked to a nucleic acid sequence encoding a
substrate
trapping mutant protein tyrosine phosphatase in which the wildtype protein
tyrosine
phosphatase catalytic domain invariant aspattate residue is replaced with an
amino acid
1 ~ which does not cause si~ificant alteration of the Km of the enzyme but
which results in
a reduction in Kcat to less than 1 per minute. and in which at least one
wildtype protein
tyrosine phosphatase tyrosine residue is replaced with an amino acid that is
not capable
of being phosphorylated. In certain embodiments, the promoter may be a
regulated
promoter as provided he:ein. for example a tetracylc:ne-repressible promoter.
In certain
embodiments the recombinant expression construct is a recombinant viral
expression
construct as provided herein. Examples of mammalian expression systems include
the
COS-7 lines of monkey kidney fibroblasts. described by Gluzman, Cell ?3:175
(1981),
and other cell lines capable of e.~cpressing a compatible vector. for example,
the C1?7,
3T3. CHO. HeLa and BHK cell lines. Mammalian expression vectors will comprise
an
?5 origin of replication. a suitable promoter and enhancer. and also any
necessary ribosome
binding sites. polvadenvlation site. splice donor and acceptor sites.
transcriptional
termination sequences. 'and ~' l3aukin Q nontranscribed sequences. for example
as
desc:ibed he:ein regarding the preparation of PTP zxpression constructs. DVS
sequences derived from the SV:~O splice. and polyadenylation sites may be used
to
;0 provide the required nontranscribed genetic dements. Introduction of the
construct into
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
the host cell can be effected by a variety of methods with which those skilled
in the art
will be familiar. including but not limited to. for eaampie, calcium phosphate
transfection. DE.aE-De:ctran mediated transfection. or eiectroporation (Davis
et aL.
1986 Basic _Llerhods in :Llolecular Biolo~)-
Identification of nucleic acid molecules for use as antisense agents.
which includes antisense oligonucleotides and ribozymes specific for nucleic
acid
sequences encoding PTPs (including subsu'ate ~ppmg mutant PTPs) or variants or
dents thereof and of DNA oligonucleotides encoding PTP genes (including
substrate trapping mutant PTPs) for targeted delivery for genetic therapy.
involve
methods well known in the art- For e:cample, the desirable properties. lengths
and other
characteristics of such oligonucieotides are well known. In certain preferred
embodiments such an antisense oligonucleotide comprises at least 1 ~
consecutive
nucleotides complementary to an isolated nucleic acid molecule encoding a
substrate
trapping mutant PTP as provided herein. Antisense oligonucleotides are
typically
designed to resist degradation by endogenous nucieolytic enzymes by using such
linkages as: phosphorothioate. methylphosphonate, sulfone, sulfate, ketyl.
phosphorodithioate, phosphorattudate, phosphate esters. and other such
linkages (see.
e.g., Agrwal et al., Tetrehedron Lett. ?8:559-X542 (1987); Miller et al., .I.
:gym. Chem.
Soc: 93:6657-6665 (1971): Stec et al.. Tetrehedron Lett. ?6:?191-2194 (1985);
Moody
ZO et al., ~Vucl. .-Icidr Res. I ?:769-~78~ ( 1989); Uzaanski et al., ~Vucl.
.=lcids Res. ( 1989);
Letsinger et al., Tetrahedron X0:137-1=~S (1984); Eckstein. dnnu. Rev.
Biochem.
5:367-:~02 (1985); Eckstein- Trends Biol. Sci. I-x:97-100 (1989); Stein In:
Oligodeoryrrucleotides. ,~ntisense Inhibitors of Gene Expression. Cohere, Ed.
Macmillan
Press, London. pp. 97-117 (1989); Japer et al.. Biochemistry 2; :7237-7246
(1988)).
Antisense nucleotides are oligonucleotides that bind in a sequence-
specific manner to nucleic acids_ such as mR:~t~ or DNA. When bound to mR'~A
that
has complementary sequences. antisense prevents translation of the mR.'~1A
(see. e.,;..
U.S. Patent ~lo. p_168,0>; to ~ltman et al.: U.S. Patent Rio. 5.190.951 to
Inouve. U.S_
Patent ~o. 5.1>j.917 to Burch: LT.S. Patent Vo. 5.087.617 to Smith and Clusel
et al.
30 (1990 .Vucl. .~c:dr Res. 5!:5-10=-~-X11- which describes dumbbell antisense
CA 02375145 2001-11-23
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;5
oligonucleotides). Triplex molecules refer to single DNA strands that bind
duplex
DNA forming a colinear triplex molecule, thereby preventing transcription
(see, e.,;..
U.S: Patent No. x.176.996 to Hogan et al., which describes methods for making
synthetic oligonucleotides that bind to target sites on duplex DNA).
According to this embodiment of the invention, particularly useful
antisense nucleotides and triple:c molecules are molecules that are
complementary to or
bind the sense strand of DNA or mRNA that encodes a PTP polypeptide (including
substrate trapping mutant PTPs), such that inhibition of translation of
mR:'~1A encoding
the PTP polypeptide is erected.
A ribozyme is an RNA molecule that specifically cleaves RNA
substrates, such as mR'VA. resulting m specific inhibition or interference
with cellular
gene expression. There are at least five known classes of ribozymes involved
in the
cleavage andlor ligation of R.~A chains. Ribozymes can be targeted to any
R:~iA
transcript and can catalytically cleave such transcripts (see, e.g., U.S.
Patent No.
S.?72,262; U.S. Patent No. ~.1=x.019; and U.S. Patent Nos. ~,168,05~,
x,180,818,
5,116,742 and ~.093,?46 to Cech et al.). According to certain embodiments of
the
invention. any such PTP (including substrate trapptag mutant PTP) mR.:~'~-
specific
ribozyme, or a nucleic acid encoding such a ribozyr.:e. may be delivered to a
host cell to
effect inhibition of PTP gene expression. Ribozymes. and the like may
therefore be
delivered to the host cells by DNA encoding the ribozyme linked to a
eukaryotic
promoter. such as a eukaryotic viral promoter, such that upon introduction
into the
nucleus. the ribozyme will be directly transcribed.
The expressed recombinant PTP polypeptides or fusion proteins
(including substrate trapping mutant PTPs) may be useful in intact host cells;
in intact
'_'S organelles such as cell membranes. intracellular vesicles or other
cellular organelles: or
in disrupted cell preparations including but not limited to cell homogenates
or lysates.
microsomes. uni- and muitilamellar membrane vesicles or other preparations.
Alternatively. expressed recombinant PTP polypeptides or fusion proteins can
be
recovered and purified from recombinant cell cultures by methods including
ammonium
~0 sulfate or ethanol precipitation. acid extraction. anion or canon exchange
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
36
chromatography. phosphocellulose chromatography, hydrophobic interaction
chromato°raphy, affinity chromato~aphy, hydroxylapatite chromatography
and lectin
chromatography. Protein refolding steps can be used- as necessary. in
completing
configuration of the mature protein. Finally. high performance liquid
chromatography
(HPLC) can be employed for final purification steps.
Turning to another aspect of the invention, there is provided a method of
identifying a tryosine phosphorylated protein which is a substrate of a PTP. A
''sample"
as used herein refers to a biological sample containing at least one tyrosine
phosphorylated protein. and may be provided by obtaining a blood sample.
biopsy
specimen. tissue e:cplant. organ culture or any other tissue or cell
preparation from a
subject or a biological source. A sample may further refer to a tissue or cell
preparation
in which the morphological integrity or physical state has been disrupted. for
example,
by dissection. dissociation. solubilization, fractionation- homogenization.
biochemical
or chemical earaction, pulverization, lyophilization, sonication or any other
means for
1~ processing a sample derived from a subject or biological source. In certain
preferred
embodiments, the sample is a cell lysate, and in certain particularly
preferred
embodiments the lysate is a detergent solubilized cell lysate from which
insoluble
components have been removed according to standard cell biology techniques.
The
subject or biological source may be a human or non-human animal. a primar'-'
cell
culture or culture adapted cell line including but not limited to genetically
engineered
cell lines that may contain chromosomally inte~ated or episomal recombinant
nucleic
acid sequences. immortalized or immortalizable cell lines. somatic cell hybrid
cell lines,
differentiated or differentiatable cell lines. transformed cell lines and the
like.
Optionally. in certain situations it may be desirable to treat cells in a
biological sample
with pervanadate as described herein. to enrich the sample in tyrosine
phosphorylated
proteins. Other means may also be employed to et~ect an increase in the
population of
tyrosine phosphorylated proteins present in the sample. including the use of a
subject or
biological source that is a cell line that has been transfected with at least
one gene
encoding a protein tyrosine lCinases. Additionally or alternatively. protein
tyrosine
~0 phosphoryiation may be stimulated in subject or biological source cells
using any one or
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
37
more of a variety of well known methods and compositions known in the art to
stimulate protein tyrosine kinase activity. These stimuli may include. without
limitation. exposure of cells to cytokines. ~owth factors. hormones, peptides.
small
molecule mediators or other agents that induce PTK-mediated protein tyrosine
phosphorylation. Such agents may include, for example, interleukins,
interferons.
human ~owth hormone. insulin and fibroblast ~owth factor (FGF), as well as
other
agents with which those having ordinary skill in the art will be familiar.
According to the subject invention. a sample comprising at least one
tyrosine phosphorylated protein is combined with at least one substrate
trapping mutant
IO PTP as provided herein. under conditions and for a time sufficient to
permit formation
of a complex between the tyrosine phosphorylated protein and the substrate
trapping
mutant PTP. Suitable conditions for formation of such complexes are known in
the art
and can be readily determined based on teachings provided herein. including
solution
conditions and methods for detecting the presence of a complex. Next the
presence or
absence of a complex comprising the tyrosine phosphorylated protein and the
substrate
trapping mutant PTP is determined. wherein the presence of the complex
indicates that
the tyrosine phosphorylated protein is a substrate of the PTP with which it
forms a
complex.
Substrate trapping mutant PTPs that associate in complexes with
tyrosine phosphorylated protein substrates may be identified by any of a
variety of
techniques laiown in the art for demonstrating an intermolecular interaction
between a
PTP and a PTP substrate as described above. for example, co-purification. co
preoipitation. co-immunoprecipitation_ radiometric or fluorimetric assays,
western
immunoblot analyses. amity capture including affinity techniques such as solid-
phase
~5 li~and-counteriisand sorbent techniques. amity chromatography and surface
affinity
plasmon resonance. and the like (see. e.o . LT.S. Patent ~Io. ~.3~2.660).
Determination
of the presence of a PTP!substrate complex may employ antibodies. including
monoclonal. polvcional. chimeric and single-.:hain antibodies. and the like.
that
specifically bind'to the PTP or the tyrosine phosphorylated protein substrate.
Labeled
;0 PTPs and/or Labeled tyrosine phosphoryiated substrates can also be used to
detect the
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
38
presence of a comple:c. The PTP or phosphorylated protein can be labeled by
covalently or non-covalently attaching a suitable reporter molecule or moiety.
for
e:cample any of various enzymes. fluorescent materials, luminescent materials
and
radioactive materials. E:camples of suitable enzymes include. but are not
limited to,
horseradish pero:cidase, biotin. alkaline phosphatase, ~-~alactosidase and
aceryicholinesterase. E:camples of suitable fluorescent materials include. but
are not
limited to, umbeiliferone. fluorescein, ffuorescein isothiocyanate. rhodamine.
dichlorotriazinylamine lluorescein. dansyl chloride and phycoerythrin.
Appropriate
luminescent materials include luminol, and suitable radioactive materials
include
radioactive phosphorus [''P], iodine ['''~I or'3'I] or tritium [3H].
Using such approaches, representative comple:ces of PTP 1 B with p210
bcr:abl. of PTP-PEST with p 130". of TC-PTP with Shc (e.,;.. Tiganis et al..
1998 .Llol.
Cell. Biol. 18:1622-I6~~) and of PTPH1 with pp97/VCP may be readily identified
by
western immunoblot analysis as described below. These associations may be
observed.
1 S for e.~cample, in lysates from several cell lines and in transfected
cells. indicating that
p210 bcr:abl, pi30'~, Shc and VCP represent major physiologically relevant
substrates
for PTP1B, PTP-PEST. TC-PTP and PTPH1, respectively. The compositions and
methods of the present invention. which may be used. as e:cemplified herein.
to identify
specific tyrosine phosphoryiated substrates for PTPIB. PTP-PEST and PTPHI, are
generally applicable to any member of the PTP family, including but not
limited to TC-
PTP, PTP,j, MKP-I. DEP-I. PTP~. SHP?, PTP-PEZ, PTP-v~Gl. LC-PTP, CD45,
L.4R and PTPY10.
In certain embodiments of this aspect of the invention. the sample may
comprise a cell that naturally e:cpresses the tyrosine phosphorylated protein
that is a
''S PTP substrate. while in certain other embodiments the sample may comprise
a cell that
has been transfected ~.vith one or more nucleic acid molecules encoding the
substrate
protein. For e:cample: the sample may comprise a cell or population of cells
that has
been transfected with a nucleic acid library such as a cDNA library that
contains at least
one nucleic acid molecule encoding a substrate protein. Any tyrosine
phosphorylated
protein is suitable as a potential substrate in the present invention.
Tyrosine
CA 02375145 2001-11-23
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39
phosphorylated proteins are well known in the art. Specific examples of
appropriate
substrates include. without limitation. p 1 ~0'u, pp97/VCP. the EGF receptor.
p210
bcr:abl. ~fAP kinase. Shc and the insulin receptor. Of particular interest are
tyrosine
phosphorylated proteins that have been implicated in a mammalian disease or
disorder.
According to the present invention_ substrates may include full length
tyrosine phosphoryiated proteins and polypeptides as well as fra~nents (e.o ,
portions),
derivatives or analogs thereof that can be phosphorylated at a tyrosine
residue. Such
fragments. derivatives and analogs include any PTP substrate polypeptide that
retains at
least the biological function of interacting with a PTP as provided herein.
for example
by forming a complex with a PTP. :~ fragment. derivative or analog of a PTP
substrate
polypeptide: including substrates that are fusion proteins, may be (l) one in
which one
or more of the amino acid residues are substituted with a conserved or non-
conserved
amino acid residue (preferably a conserved amino acid residue), and such
substituted
amino acid residue may or may not be one encoded by the genetic code, or (ii)
one in
1~ which one or more of the amino acid residues includes a substituent soup,
or (iii) one
in which the substrate polvpeptide is fusers with another compound. such as a
compound to increase the half life of the polypeptide (e.g.. polyethylene
glycol) or a
detectable moiety such as a reporter molecule. or (iv) one in which additional
amino
acids are fused to the substrate polypeptide. including amino acids that are
employed for
purification of the substrate polypeptide or a proprotein sequence. Such
fragments.
derivatives and analogs are deemed to be within the scope of those skilled in
the art.
The subject invention also contemplates certain embodiments wherein
the substrate trapping mutant PTP that is combined with the sample) is a
mutant PTP
that is expressed by a cell, including embodiments wherein the cell has been
transfected
with one or more nucleic acid molecules encoding the mutant PTP. Thus. the
method
of identifying a tyrosine phosphory lated protein which is a substrate of a
PTP may
include in certain embodiments combining a sample comprising a tyrosine
phosphorylated protein with a mutant PTP wherein the sample comprises a c~11
e:cpressing either or both of the tyrosine phosphorylated protein and the
mutant PTP.
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.~0
Optionally. the cell may be transfected with nucleic acids encoding either or
both of the
tyrosine phosphorylated protein and the mutant PTP.
In another aspect_ the invention provides methods of identifying an agent
that alters the interaction between a PTP and a tyrosine phosphorylated
protein that is a
substrate of the PTP. through the use of screening assays that detect the
ability of a
candidate agent to alter (l. e.. increase or decrease) such interaction. The
interaction
between the PTP and its substrate may be determined enzymatically, for
e:cample by
detecting catalytic substrate dephosphorylation. Alternatively. the
interaction between
the PTP (including a substrate trapping mutant PTP) and its substrate may be
determined as a binding interaction. and in preferred embodiments such
interaction is
manifested as detection of a comple:c formed by PTP-substrate binding,
according to
criteria described herein. Agents identified according to these methods may be
aaonists
(e.g., agents that enhance or increase the activity of the wildrype PTP) or
antagonists
(e.o , agents that inhibit or decrease the activity of the wildrype PTP) of
PTP activity.
1 ~ Agents may be identified from among naturally occurring or non-naturally
occurring
compounds. including synthetic small molecules as described below.
In certain embodimenu. wherein the screening assay is directed to PTP
catalytic activity. the tyrosine phosphorylated protein that is a substrate of
the PTP can
be identined as described above. which method features the use of a novel
substrate
trapping mutant PTP as disclosed herein. Accordingly, a PTP and a tyrosine
phosphoryiated substrate are combined in the absence and in the presence of a
candidate
agent where the substrate has first been identified as described above using a
substrate
trapping mutant PTP. The PTP and the substrate are combined under conditions
permissive for the detectable dephosphorylation of the subsnate to occur.
?S Anv suitable method may be used to detect phosphoprotein
dephosphorylation: such methods are well known in the art and include. without
limitation. detection of substrate catalysis by one or more of. e.o .
radiometric.
tluorimetric. densitometric. spectrophotometric. chromatoQraphic_
eiec:rophoretic.
colorimetric or biometric assays. T'ne level of dephosphorylatiun of the
substrate in the
absence of the agent is compared to the level ut' dephosphorylation of the
substrate in
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=~ 1
the presence of the agent, such that a difference in the level of substrate
dephosphorylation (e.g., a statistically significant increase or decrease)
indicates the
anent alters the interaction between the protein tyrosine phosphatase and the
substrate.
For instance. an znzymatic activity assay utilizing a wildtype PTP can be
carried out in the absence and presence of a candidate agent. Enzymatic
activity assays
known in the art include. for e:cample, PTP activity assays using a tyrosine
phosphorylated 'zP-labeled substrate as described in Flint et al. ( 1993 E
L1B0 f
IZ:I937-19~. A decrease in the PTP enzymatic acnnty tn the presence of the
candidate agent indicates that the agent inhibits the interaction between the
PTP and its
substrate. Conversely, an increase in PTP enzymatic acnvity tn the presence of
the
agent indicates that the agent enhances the interaction between the PTP and
its
substrate.
In certain other embodiments, wherein the screening assay is directed to
identifying an agent capable of altering a substrate trapping mutant PTP-
substrate
1 ~ binding interaction.. the substrate trapping mutant PTP (as described
herein) and a
tyrosine phosphoryiated substrate are combined in the absence and in the
presence of a
candidate agent under conditions and for a time su~cient to permit formation
of a
comple:c between the tyrosine phosphoryiated protein and the substrate
trapping mutant
PTP. thereby producing a combination. The formation of a comple:c comprising
the
tyrosine phosphorylated protein and the substrate trapping mutant protein
tyrosine
phosphatase in the combination is ne:ct determined (as also provided herein),
wherein a
difference between the level of comple:c formation (e.g.. a statistically
significant
difference) in the absence and in the presence of the agent indicates that the
agent alters
(i.e.. increases or decreases) the interaction between the protein tyrosine
phosphatase
?5 and the substrate. Altemativelv. a competitive bindins assay can be carried
out utilizing
the substrate trapping mutant'PTP in the absence and presence of a candidate
agent.
Competitive binding assays known in the art include. for e~cample. LT.S.
Patent
Rio. ~.~ ~=.660. which desc:ibes methods suitable for use according to these
embodiments of the present invention. ~ decrease in the e:ctent of PTP-
substrate
~0 binding in the presence of the anent to be tested indicates that the went
inhibits the
CA 02375145 2001-11-23
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4''
interaction between the PTP and iu substrate. Conversely, an increase in the
extent of
binding in the presence of the agent to be tested indicates that the agent
enhances the
interaction between the PTP and iu substrate.
Candidate menu for use in a method of screening for an agent that alters
the interaction between a PTP and its tyrosine phosphorylated protein
substrate
according to the present invention may be provided as "libraries'' or
collections of
compounds. compositions or molecules. Candidate agents that may interact with
one or
more PTPs (including agents that interact with a substrate trapping mutant PTP
as
provided herein) may include members of phosphotvrosyl peptide libraries as
described
in Songyang et al. (1995 Vatz~re 373:>>6->j9; 1993 Cell 7?:767-778) that bind
to the
PTP. Peptides identified from such peptide libraries can then be assessed to
determine
whether tyrosine phosphorylated proteins containing these peptides e:cist in
nature.
Alternatively, libraries of candidate molecules to be screened may typically
include
compounds known in the art as °small molecules" and having molecular
weighu less
1~ than 10' daltons. preferably less than 10' daltons and still more
preferably less than 10'
daltons. For e:cample, members of a library of test compounds can be
administered to a
plurality of samples, each containing at least one substrate trapping mutant
PTP and at
least one tyrosine phosphorylated protein that is a substrate of the PTP as
provided
herein, and then assayed for their ability to enhance cr inhibit mutant PTP
binding to
ZO the substrate. Compounds so identified as capable of altering PTP-substrate
interaction
(e.g.. binding andlor substrate phosphotyrosine dephosphorylation) are
valuable for
therapeutic and/or dia~ostic purposes, since they permit treatment and/or
detection of
diseases associated with PTP activity. Such compounds are also valuable in
research
directed to molecular si~aling mechanisms that involve PTPs, and to
refinements in
25 the discovery and development of future compounds e:chibiting heater
specificity.
Candidate agents further may be provided as members of a combinatorial
library. which o_ refe:ablv includes synthetic agents prepared according to a
plurality of
predetermined chemical reactions performed in a plurality of reaction vessels.
For
e:cample. various startins compounds may be prepared employing one or more of
solid-
30 phase synthesis. recorded random mi.~c methodologies and recorded reaction
split
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
techniques that permit a liven constituent to traceably undergo a plurality of
permutations and/or combinations of reaction conditions. The resulting
products
comprise a library that can be screened followed by iterative selection and
synthesis
procedures. such as a synthetic combinatorial library of peptides (see e.,;..
PCT~'LrS9Ii0869~. PCTIUS9110~666. which are hereby incorporated by reference
in
their entireties) or other compositions that may include small molecules as
provided
herein (see e.o , PCTlLJS94/085~2. EP 0'rl~ U.S. 5.798,035. U.S. 5,789,172,
U.S.
5,751,629, which are hereby incorporated by reference in their entireties).
Those
having ordinary skill in the art will appreciate that a diverse assortment of
such libraries
may be prepared according to established procedures, and tested using
substrate
trapping mutant PTPs according to the present disclosure.
The invention also pertains to a method of reducing the activity of a
tyrosine phosphorylated protein. comprising administering to a subject a
substrate
trapping mutant PTP in which (i) the wildtype PTP catalytic domain invariant
aspartate
residue is replaced with an amino acid which does not cause significant
alteration of the
Km of the enzyme but which results in a reduction in Kcat to less than 1 per
minute
(less than 1 miri') (e.o . an alanine residue), and (ii) at least one wildtype
tyrosine
residue is replaced with an amino acid that is not capable of being
phosphorylated.
whereby interaction of the substrate trapping mutant protein tyrosine
phosphatase with
the tyrosine phosphorylated protein reduces the activity of the tyrosine
phosphorylated
protein. In certain preferred embodiments. the tyrosine phosphorylated protein
is VCP,
p130"~, the EGF receptor. p210 ber:abl. ~ Vie- Shc or the insulin receptor. In
certain other preferred embodiments. the protein tyrosine phosphatase is PTP 1
B, PTP
PEST. PTP;~, VIKP-l. DEP-1, PTPu. PTP.'C1. PTPYIO. SHP''. PTP-PEZ. PTP-VtEGl.
35 LC-PTP, TC-PTP, CD45. L~R or PTPHI.
Without wishing to be bound by theory. such a mutant PTP may reduce
the activity of the corresponding wildtype PTP by forming a complex with the
tyrosine
phosphorylated protein substrate of the wildtype PTP. thereby rendering the
substrate
unavailable for catalytic deahosphorylation by the wildrype enzyme. The
substrate
trapping mutant PTP thus binds to the phosphoprotein substrate without
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
dephosphorylating it (or catalyzing dephosphorylation at a neatly reduced
rate). thereby
blocking the activity of the dephosphorylated protein substrate and reducing
its
downstream effects. :~s used herein. "reducing" includes both reduction and
complete
abolishment of one or more activities or functions of the phosphorylated
protein
substrate.
In one aspect of the method of reducing the activity of a tyrosine
phosphoryiated protein. a method is provided for reducing the transforming
effects of at
least one oncogene associated with phosphorylation of p130'~, a substrate of
PTP-
PEST. The method Generally comprises administering to a subject a substrate
trapping
mutant PTP-PEST in which the wildtvpe PTP catalytic domain invariant aspartate
residue is replaced with an alanine residue, and in which at least one
wildtvpe tyrosine
residue is replaced with an amino acid that is not capable of being
phosphorylated.
Whereas wildtype PTP-PEST binds and dephosphorylates the substrate p130"5,
thereby
negatively regulating this substrate's downstream biological effects, the
subject
1~ invention substrate trapping PTP-PEST mutants bind but cannot
dephosphorylate
p 1;0"~ (or do so at a ~eatiy reduced rate). According to the non-limiting
theory
disclosed above. the substrate is thus sequestered in the complex with the
substrate
trapping PTP-PEST and cannot e.~cert its downstream effects. In certain
embodiments of
this method, the oncogene may be one of v-crk, v-5re or c-Ha ras.
Similarly. the invention relates to a method of reducing the formation of
signaling complexes associated with p130"', particularly those signaling
complexes
which induce mitogenic pathways, comprising administering to a mammal
substrate
trapping mutant PTP-PEST as provided above. The PTP binds to andlor
dephosphorylates p1~0'~. thereby negatively regulating the downsue3m effects
of
~5 p130'_- and reducing the formation of signaling complexes associated with
p1~0"s. ~s
another example. in certain embodiments the invention relates to regulation of
the cell
cycle by the PTPHl substrate pp97/VCP. wherein a substrate trapping mutant
PTPH1
as provided herein ( l. e.. a double mutant that is caralytically attenuated
and in which a
wildtype tyrosine has been replacedl can alter the interaction between PTPHI
and VCP.
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
~s provided herein. the substrate trapping mutant PTPs of the present
invention may be useful in virtually any situation where biological regulation
involving
PTP-regulated signal transduction is involved, for example. in place of. or in
addition
to, a corresponding wildtvpe PTP. T'ne advantages of such utility of the
subject
invention lie in the ability of a substrate trapping mutant PTP to mimic the
function of
its corresponding wildtype enzyme. e.o - to impair the biological signaling
activity of a
tyrosine phosphorylated substraxe subsequent to dephosphorylation mediated by
wildtype PTP, without inducing the harmful cytotoxic effects commonly observed
when
wildtype PTP is administered and/or overexpressed. Thus. the invention also
pertains
to a method of reducing the cytotoxic effects associated with administration
or
overexpression of wild type PTPs. For example. CS mutants of VIKP-1 have been
shown to have the same functional effect as wild type VIKP-I without induction
of
potentially harmful side effects. Thus. PTPs described herein. in which the
wildtype
PTP catalytic domain invariant aspartate residue is replaced with an amino
acid which
1 ~ does not cause significant alteration of the Km of the enzyme but which
results in a
reduction in Kcat to less than 1 per minute (less than 1 min') (e.g., an
alanine residue),
and in which at least one wildtype tyrosine residue is replaced with an amino
acid that is
not capable of being phosphorylated. can in many situations be substituted for
a
counterpart wildtype enzyme, where such a counterpart wildtype enzyme can
specifically interact with the same substrate as the mutant PTP.
The substrate trapping mutant PTPs described herein may also be used
therapeutically to alter (l. e.. increase or decrease) the activity of a
tyrosine
phosphorylated protein, such as by a gene therapy method in which a nucleic
acid. for
example. a recombinant expression construct as described above, encoding a
substrate
trapping mutant PTP (or a functional portion thereof) which retains the
ability to bind to
its tyrosine phosphorylated substrate. is introduced into a subject and is
expressed. The
mutant PTP replaces. either partially or totally. a corresponding host PTP
enzyme that is
normally produced in the subject or may compete with the host PTP for binding
to the
substrate. For example. where a specinc tyrosine phosphorylated protein
substrate may
~0 be implicated in a particular disease or disorder. at least one PTP capable
of
CA 02375145 2001-11-23
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46
dephosphorylating the suspect substrate may be identified. A corresponding
substrate
trapping mutant PTP can be administered either directly or by gene therapy.
using the
compositions and methods described herein. Such a mutant PTP may sequester the
tyrosine phosphorylated substrate, thereby inhibiting or reducing the
substrates role in
the disease process. In a preferred embodiment, the substrate trapping mutant
PTP of
the present disclosure is administered in place of a corresponding wildtype
enzyme. tn
order to reduce the cytotoxic effects associated with overe:cpression of the
wild type
enzyme. Procedures for geae therapy are known in the art (see, e.g., U.S.
Patent No.
5,399.36) and can be modified by known methods known in order. to express the
I O subject invention substrate trapping mutant PTPs.
The methods of the present invention are specifically e:cemplified herein
with respect to the phosphatases PTPHl. PTPIB and PTP-PEST: however. it is
understood that the invention is not limited to these specific PTPs but is
applicable to
all members of the PTP family. In order to identify potential substrates of
PTPHI,
I~ PTPIB and PTP-PEST. mutant (i.e., altered or substrate trapping) forms of
PTPH1,
PTP I B and PTP-PEST are generated as described herein that are catalytically
attenuated but that retain the ability to bind substrates.
In certain embodiments. the invention relates in part to PTP 1 B(D 13 I r1),
in which the aspartate residue at position 131 of wildtype PTPIB is repiaeed
with
20 alanine. and in which fiuther a PTP tyrosine residue may optionally be
replaced with a
non-phosphorylatable residue. In certain other embodiments the invention
relates to the
phosphatase PTP-PEST(D 199A) and in certain other embodiments to PTP
PEST(C231 S), which in either case may further have a PTP tyrosine residue
optionally
replaced with a non-phosphorylatable residue. In particularly preferred
embodiments
25 the invention relates to PTPH1(Y676F~'D8I I~).
As noted above. in certain embodiments the invention relates . to a
substrate trappins mutant PTP-PEST. PTP-PEST is an 86 kDa cvtosolic PTP
(Chartist
et al.. 199 Biochem. .I 308:~'_'~--~~~: den Hertog et al., 1992 Biochem.
Biophys. Res.
Commzrn. 18-~:1~'-Il-1==~9: Take!cawa et al.. 199? Biocnem. Biopnys. Res.
Commun. 189'.
30 1='_'~-1'_'30: Yang et al.. 1993 J. Biol. Chem. ?68:66=''-6623: Yang et
a1._ l99? J. Biol.
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
~7
Chem. Z68: I 7 660) which is e:~pressed ubiquitously in mammalian tissues (Yi
et al..
1991 Blood -8:?''2?-??~8), and which e:chibits hi°h specific activity
when assayed in
vitro using artificial tyrosine phosphorylated substrates (Garton and Tonks,
199 E:LIBO
.I. 13:3763-3771). PTP-PEST is subject to regulation via phosphorylation of
Ser39 in
vitro and in vivo. This modification is catalyzed by both protein kinase C
(PKC) and
protein kinase A (PK~), and results in reduced PTP-PEST enzyme activity due to
an
increase in the Km for the dephosphorylation reaction catalyzed by this PTP
(Garton
and Tonks, 1994 E:LIBO ,I. 13:3763-3771). Additional intracellular regulatory
mechanisms may include PTP-PEST-mediated dephosphorylation of one or more
cytosolic substrates of tyrosine kinases.
As disclosed herein and described in the Examples. the substrate
specificities of PTP 1 B and of PTPHl may be characterized by methods that
relate to
PTP catalytic and/or binding interactions with substrate. e.o .
dephosphorylation and
substrate trapping in vitro and in vivo. PTP1B (see. e.o.. Barford et al..
1994 Science
263:1397; Jia et al., 1996 Science ?68:1750 and PTPH1 (see. e.o . U.S. Patent
Nos.
6.696,911 and 6,863,781) are well known in the art. The substrate trapping
methods
provided herein are generally applicable to any PTP by virtue of the invariant
PTP
catalytic domain aspartate residue and the frequency of tyrosine in PTP amino
acid
sequences. and should therefore prove useful in delineating the substrate
preferences of
other PTP family members. In particular. the use of mutant catalvtically
impaired
PTPs to trap. and thereby isolate. potential substrates permits the
identification of
physiologically important substrates for individual PTPs, leading to improved
understanding of the roles of these enzymes in revelation of cellular
processes_
Furthermore. replacement of PTP tyrosine residues with amino acids that cannot
be
26 phosphorylated provides substrate trapping mutant PTPs that are not
impaired in their
ability to interact with tyrosine phosphorylated protein substrates.
The present invention also pertains to pharmaceutical compositions
comprising a substrate trapping mutant PTP in which (l) the wildtype PTP
catalytic
domain invariant aspartate residue is replaced with an amino acid which does
not cause
~0 si~incant alteration of the Km of the enzyme but which results in a
reduction in Kcat
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
48
to less than 1 per minute (less than 1 miri') (e.o , an alanine residue); and
(ii) at least
one wildtype tyrosine residue is replaced with an amino acid that is not
capable of being
phosphoryiated (e.,;., not seine or threonine_ nor any other naturally
occurring or non-
natuallv occurring amino that may be phosphorylated). The PTP of the preseat
invention may therefore be formulated with a physiologically acceptable medium
such
as, for e:cample, a pharmaceutically acceptable carrier or diluent, to prepare
a
pharmaceutical composition.
For administration to a patient one or more polypeptides (including
substrate trapping mutant PTPs), nucleic acid molecules (including recombinant
e:cpression constructs encoding substrate trapping mutant PTPs) and/or
modulating
agents (including agents that interact with a PTP and/or a substrate trapping
mutant
PTP) are generally formulated as a pharmaceutical composition. :~
pharmaceutical
composition may be a sterile aqueous or non-aqueous solution, suspension or
emulsion.
which additionally comprises a physiologically acceptable carrier (i.e., a non-
toxic
1 ~ material that does not interfere with the activity of the active
ingredient). S uch
compositions may be in the form of a solid, liquid or gas (aerosol).
Alternatively,
compositions of the present invention may be formulated as a lyophilizate or
compounds may be encapsulated within liposomes using well known technology.
Pharmaceutical compositions within the scope of the present invention may also
contain
other components. which may be biologically active or inactive. Such
components
include. but are not limited to. buffers (e.o , neutral buffered saline or
phosphate
buffered saline), carbohydrates (e.o , glucose. tnannose, sucrose or
de:ctrans), mannitol.
proteins. polypeptides or amino acids such as glycine_ antio~cidants,
cheiating agent
such as EDT: or glutathione. stabilizers. dyes. flavoring agents. and
suspending agents
and/or preservatives.
Any suitable carrier known to those of ordinary skill in the art may be
employed in the pharmaceutical compositions of the present invention. Carriers
for
therapeutic use are well known. and are described_ for e:cample. in Remingtons
Pharmacezuical Sciences. Vtack Publishing Co. (A.R. Gennaro ed. 1980. In
<leneral.
the type of carrier is selected based on the mode of administration.
Pharmaceutical
CA 02375145 2001-11-23
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19
compositions may be formulated for any appropriate manner of administration.
including, for example, topical. oral. nasal. intraocular, intrathecal,
rectal, vaginal,
sublingual or parenteral administration- including subcutaneous. intravenous.
intramuscular. intrasternal. intracavernous. intrameatal or intraurethral
injection or
infiision. For parenteral administration. the carrier preferably comprises
water, saline.
alcohol. a fat a wax or a buffer. For oral administration, any of the above
carriers or a
solid carrier. such as mannitol, lactose, starch, magnesium stearate, sodium
saccharine.
talcum. cellulose, kaolin. glycerin. starch dextrins, sodium alginate,
' carboxymethylcellulose, ethyl cellulose. glucose, sucrose andlor magnesium
carbonate.
may be employed.
~, pharmaceutical composition (e.o . for oral administration or delivery
by injection) may be in the form of a liquid (e.o.. an eli.~cir, syrup.
solution. zmulsion or
suspension). ..liquid pharmaceutical composition may include, for example. one
or
more of the following: sterile diluents such as water for injection- saline
solution,
1 S preferably physiological saline, Ringer's solution. isotonic sodium
chloride. tLYed oils
such as synthetic mono or diglycerides which may serve as the solvent or
suspending
medium. ' polyethylene glycols. glycerin. propylene glycol or other solvents;
antibacterial agents such as benzyi alcohol or methyl paraben; antioxidants
such as
ascorbic acid or sodium bisulfite: chelating agents such as
ethylenediaminetetraacetic
acid; bui~ers such as acetates, citrates or phosphates and agents for the
adjustment of
tonicity such as sodium chloride or dextrose. A parenteral preparation can be
enclosed
in ampoules. disposable syringes or multiple dose vials made of Mass or
plastic. The
use of physiological saline is preferred- and an ~jec~le pharmaceutical
composition is
preferably sterile.
The compositions desi.ibed herein may be formulated for sustained
release (l. e.. a formulation such as a capsule or sponge that erects a slow
release of
compound following administration). Such compositions may generally be
prepared
using well known technology and administered by. for example. oral- rectal or
subcutaneous implantation. or by implantation at the desired target site.
Sustained-
~0 release formulations may contain an agent dispersed in a carrier matrix
and~'or contained
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
within a reservoir surrounded by a rate controlling membrane. Carriers for use
within
such formulations are biocompatible. and may also be biode3adable: preferably
the
formulation provides a relatively constant level of active component release.
The
amount of active compound contained within a sustained release formulation
depends
upon the site of implantation, the rate and expected duration of release and
the nature of
the condition to be treated or prevented.
For pharmaceutical compositions comprising a nucleic acid molecule
encoding a substrate trapping mutant PTP polypeptide (such that the
polypeptide is
generated in situ), the nucleic acid molecule may be present within any of a
variety of
delivery systems known to those of ordinary skill in the art, including
nucleic acid, and
bacterial. viral and mammalian expression systems such as, for example,
recombinant
expression constructs as provided herein. Techniques for incorporating DNA
into such
expression systems are well known to those of ordinary skill in the art. The
DNA may
also be "naked," as described. for example. in Ulmer et al., Science Z59:174~-
1749.
I~ 1993 and reviewed by Cohen, Science h9:1691-1692, 1993. The uptake of naked
DNA may be increased by coating the DNA onto biode~adable beads, which are
efficiently transported into the cells.
Within a pharmaceutical composition. a substrate trapping mutant PTP
polypeptide, a substrate trapping mutant PTP-encoding nucleic acid molecule or
a
modulating agent may be linked to any of a variety of compounds. For example.
such a
polypeptide. nucleic acid molecule or agent may be linked to a targeting
moiety (e.o , a
monoclonal or polycional antibody, a protein or a Iiposome) that facilitates
the delivery
of the agent to the target site. As used herein. a "targeting moiety" may be
any
substance (such as a compound or cell) which, when linked to an agent enhances
the
?5 transport of the agent to a target cell or tissue. thereby increasing the
local concentration
of the agent_ Targeting moieties include antibodies or fra~nents thereof.
receptors.
lisands and other molecules that bind to cells of. or in the vicinity of. the
target tissue.
An antibody targeting agent may be an intact (whole) molecule. a fragment
thereof. or a
functional ecuivalent thereof. Examples of antibody fra~nents are F(ab')2. -
Fab'. Fab
and F[v] fragments. which may be produced by conventional methods or by
genetic or
CA 02375145 2001-11-23
WO 00/75339 ~ 1 PCT/US00/14211
protein engineering. Linkage is generally covalent and may be achieved by. for
e:cample, direct condensation or other reactions. or by way of bi- or mufti-
~nctional
linkers. Targeting moieties may be selected based on the cells) or tissues) at
which
the agent is e:cpected to e:cezt a therapeutic benefit.
Pharmaceutical compositions may be administered in a manner
appropriate to the disease to be treated (or prevented). An appropriate dosage
and a
suitable duration and frequency of administration will be determined by such
factors as
the condition of the patient, the type and severity of the patient's disease,
the particular
form of the active ingredient and the method of administration. In general. an
appropriate dosage and treatment regimen provides the agents) in an amount
sufficient
to provide therapeutic andlor prophylactic benefit (e.,;., an improved
clinical outcome.
such as more frequent complete or partial remissions. or loner disease-free
and/or
overall survival). For prophylactic use, a dose should be sufficient to
prevent, delay the
onset of or diminish the severity of a disease associated with a defect in
cell si~aling,
1~ for e:cample a defect leading to abnormal cell cycle regulation.
proliferation. activation,
differentiation, senescence. apoptosis, adhesion, metabolic activity. gene
e:cpression or
the like_
Optimal dosages may generally be determined using e:cperimental
models and/or clinical trials. In general- the amount or polypeptide present
in a dose. or
produced in .nine by DNA present in a dose, ranges from about 0.01 p.g to
about I00 ug
per kg of host typically from about O.I ug to about 10 fig. The use of the
minimum
dosage that is sufficient to provide effective therapy is usually preferred.
Patients may
?enerally be monitored for therapeutic or prophylactic effectiveness using
assays
suitable for the condition being treated or prevented, which will be familiar
to those
~5 having ordinary skill in the art. Suitable dose sizes will vary with the
size of the patient.
but will typically range from about 1 mL to about X00 mL for a 10-60 kg
subject.
The following E.oamples are oiTered for the purpose of illustrating the
present invention and are not to be construed to limit the scope of this
invention. The
teachinUs of all references cited herein are hereby incorporated by reference
in their
;0 entirety.
CA 02375145 2001-11-23
WO 00/75339 ~ PCT/US00/14211
E'~A,~iPLES
E:WIPLE 1
GENER.~TION. E.'CPRESSION .aND PL:R1FIC.aTION OF ~IL;T.~NT PTP PROTEINS
Plasmid isolation. production of competent cells, transformation and
related manipulations for the cloning, amplification- construction of
recombinant
plasmids, inserts and vectors. sequencing and the like. were carried out
according to
published procedures (Sambrook et al., :Llolecular Cloning, a Laboratory
lLlanual, Cold
Spring Harbor Laboratory Press. Cold Spring Harbor, VY, 1989; Ausubel et al.,
1993
Current Protocols in .Llolecular Biology. Greene Publ. Assoc. Inc. & John
Wiley &
Sons. Inc., Boston. vIA). Recombinant nucleic acid expression constructs
encoding
human PTP-PEST (Garton et al., 1994 EyIBO J. 13:3763; Garton et al. 1996
:Llol. Cell.
Biol: 16:6408) and human PTP-1B (Brown-Shinier et al.. 1990 Proc. Nat. Acad.
Sci.
USA 87:~ 148) were prepared as described.
Point mutations within the catalytic domains of PTPs were introduced
I ~ using standard procedures. for e~cample. the invariant aspattate (D) at
amino acid
position 199 in PTP-PEST being convened to alanine (A) by a substitution
mutation
(D I99A). Thus. mutations diving rise to PTP-PEST(D199A), PTP-PEST(C?3 I S),
PTP I B(D 131 A) and PTP 1 B( C? 1 ~ S) were introduced by site-directed
mutagenesis
using the Vtuta GeneT''' in vitro mutagenesis kit (Bio-Rad, Richmond. CA)
according to
the manufacturer' s instructions. Regions containing the specified point
mutation were
they e:cchanged with the corresponding wild type sequences within appropriate
e:cpression vectors. and the replaced mutant regions were sequenced in their
entirety to
verify the absence of additional mutations.
Full length PTP-PEST proteins (wild type and mutant proteins.
containinE either .asp 199 to .-Vila. or Cys231 to Ser mutations) and the wild
type PTP-
PEST catalytic domain (amino acids 1-30~) were e:cpressed in St~9 cells using
recombinant baculovirus (BaculoGoldT-'". Phatmingen. San Die=o. C A). and
purified a.s
described in Garton and Tonks (E.LIBO J. 1~:3 X63-3771. 1994). Truncated iotms
of
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
>;
wild type and mutant PTP-PEST proteins. comprising amino acid residues 1-305
of
PTP-PEST were also expressed in E. coli as GST fusion proteins following
subcioning
of PTP-PEST DNA, in-frame downstream of GST in pG~ vectors (Pharmacia Biotech
Inc.. Uppsala. Sweden). Twenty-five ml of E: coli transformed with the
appropriate
vector were down to log Phase (ODboo aPpr°ely 0.~). Fusion protein
expression
was then induced by addition of 0.2 tnlVl isopropyl-1-thio-{3-D-
galactopyranoside. and
the cells were down for 2--~ hours at ~0°C. Cells were harvested by
centrifugation,
incubated with ~0 ~glml lysozyme in 3 ml buffer containing ~0 rt>1VI Tris-HC 1
> pH 7.-t,
5 rtuVt EDT, 1 mlVt P1~ISF, 1 m~l~t benzamid~e, ~ mg/ml leupeptin, ~ mglml
aprotinin.
0.1% Triton X-100 and 1~0 mVI NaCl, then lysed by sonication (3 Y 10s).
Following
removal of insoluble material by centrifugation (20 minutes at 300,000 Y ~),
fusion
proteins were isolated by incubation for 30 min at ~°C with 100 ml
glutathione-
SepharoseT'~ beads (Phatmacia Biotech Inc.. Uppsala, Sweden), and the beads
were then
collected by centrifugation and washed three times with Buffer A (20 mlVi Tris-
HC1,
1 ~ pH 7.-t> 1 muI EDT:. 1 muI benzamidine. 1 mglml leupeptin, 1 mglml
aprotinin. 10%
glycerol, 1% Triton X-100 and 100 mVt NaCI). This procedure yielded
essentially
homogeneous fusion protein at a concentration of 1 mg proteinlml ~utathione
Sepharose beads. PTP 1 B proteins (wild type and mutant forms) comprising
amino
acids 1-X21 were expressed in E. coli and purified to homogeneity as described
in
Barford et al. (.l. :Llol. Biol. Z39:T_'6-'730 (1994)).
~1~IPLE 2
REGULATION OF PTP1B E.'CPRESSION LEVELS BY P210 BCR:ABL
Chronic myelogenous leukemia (CVIL) is a clonal disorder of the
hematopoietic stem cell that is characterized by the Philadelphia chromosome
(Ph), in
?5 which the c-:lbl proto-oncogene on chromosome 9. encoding a protein
tyrosine kinase
(PTK~. becomes linked to the bcr Qene on chromosome ''~. This results in the
generation of a bcr:abl fusion protein. p?10 bcr:abl in which the PTK activity
is
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
enhanced relative to that of c-Abl. This e:cample demonstrates that
phosphorylation
competent p210 bcr:abl protein specifically induces PTP1B e:cpression.
When BaF; cells (lain et al.. 1996 Blood 88:1542) expressing a
temperature-sensitive mutant form of p210 bcr:abl were shifred to the
permissive
temperature for e:cpression of p210 having PTK activity, PTP 1 B mRI~A and
protein
e:cpression levels were observed to increase within 12-24 hours. coincident
with the
appearance of the active form of the PTK (see, e.o . w098/0471?; LaiVlontagne
et al.,
1998 :Llol. Cell. Biol. 18:?965). The increase in e:~pression of PTP1B was
also
observed in Philadelphia chromosome-positive (Ph'-. ) B-lymphoid cells derived
from a
C:~. patient relative to Ph- cells from the same patient. Changes in PTP 1 B
activity.
which were commensurate with the charge in enzyme protein levels. were also
observed. These changes were specific for PTP1B and were not seen in the
closely
related homologue TC-PTP (which shay 6~% ~o acid sequence identity with
PTP1B) or in other tested PTPs. including SHP-1. SHP-2 and PTP-PEST. The
specificity of PTP1B induction by p210 bcr:abl PTK activity was confirmed
»sing
kinase-detective Ratl cells (Pender?ast et al.. 1993 Cell 75:175). These cells
e:cpress an
inactive form of p210 -bcrabl_ which contains an ar°inine instead of a
lysine residue at
amino acid position 1172 and which lacks PTK activity. E.~tpression of this
p210
mutant in Ratl cells failed to result in altered PTP1B expression levels.
E,yIPLE 3
P~ 10 BCR:.aBL BINDING SUBSTR.aTE 1NTER~.CTIONS WITH a SLBSTR.aTE TR~.PPtNG
PTP
VILT:~, iT
'This e:cample describes e:cploitation of substrate interacting properties of
a substrate trapping mutant PTP to identify a PTP substrate. Substrate
trappin_ PTP
?5 polypeptides and fusion proteins were prepared as desc:ibed in E:cample 1.
Substrate trapping mutant PTP polvpeptides or fusion proteins were
contacted with lvsates derived from various cell lines. Briefly. as starring
material for
cell lysates. HeLa and COS cells were grown in Dulbecco's modified Ea=le's
medium
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
(DMEVI), containing ~% fetal bovine serum (FBS); Ratl. Wi38. C2C12 and VIvLu
cells were down in DME1~L containing 10% FBS; 293 cells were grown in D1~IE1~I
containing 10% calf senun: MCF'.OA cells were grown in o0°,'°
DMEVI. ~0°,'° Ham's F-
1'_' containing ~% hone serum.'_0 ng/ml epidermal gr°~h factor. 10
mglml insulin, 0-~
mgiml hydrocortisone and 0.?5 mgiml fun°-_izone; BaF~ cells were
maintained as
described (Jain et al., 1996 Blood 88:1 ~~2). All media also contained
penicillin and
streptomycin at 100 U/ml and 100 mg/ml_ respectively, and all cells were 'own
at
;7°C. Calcium phosphate-mediated transfection was used to introduce
cDNA encoding
wild type and mutant PTP-PEST proteins into COS cells_ These were encoded by
PTP-
PEST cDNA (Garton et al., 1996 Viol. Cell. Biol. 16:6~08) subcloned into the
plasmid
pMT2 (Sambrook et al., ~Llolecular Cloning, a Laboratory :Llarruah Cold Spring
Harbor
Laboratory Press. Cold Spring Harbor. V~'I. 1989) from which expression was
driven by
an adenovirus .major late promoter, 20 ug DN ~ was used for transfection of
each 10 cm
plate of cells. The level of expression of PTP-PEST constructs was similar in
all cases.
1~ Prior to cell lysis, 70-90% coniZuent cell cultures were treated for 30
minutes in medium containing 0.1 mVI o:cidized vanadate (pervanadate) (20 ~1
of a
fresh solution containing ~0 ttllVl sodium metavanadate (NaVO;) and ~0 mVI
H,O=
added to 10 ml culture medium). Treatment of cells with H,O, and vanadate
leads to a
synergistic increase in phosphotyrosine levels. presumably due to inhibition
of
intracellular PTPs by vanadate (Heffetz et aL_ 1990 J. Biol. Chem. ?6~:?896-
290?).
Pezvanadate treatment resulted in the appearance of at least ~0 prominent
phosphotvrosine protein bands in all cell types. whereas untreated cells
contained
virtually undetectable levels of phosphotyrosine.
Cells were lysed in Buffer A (see Example 1) containing ~ mlVl
iodoacetic acid. Following incubation at ~°C for 30 minutes. DTT was
added to
achieve a final concentration of 10 midi. Insoluble material was then removed
by
centrifusation for 20 minutes at 300.000 a e. The resultant lysates were
stable with
regard to their phosphotyrosine content during long term (several months)
storage at
-70°C and during prolonged (at least 20 hours) incubation at -
~°C_ in the absence of
exogenous added PTPs.
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
~6
Pervanadate-treated Hei.a cell lysate was fractionated by anion eYChange
chromato~aphy using a Vlono Q FPLC column (Pharmacia). The sample (~0 mg total
protein at 3 ma/ml in buffer A) was diluted in three volumes of buffer B (?0
rrWi tris-
HC 1. pH 7.-~. 1 mVt EDT. 1 mVt benzamidine. 1 mJml leupeptin, 1 mglml
aprotinin
and 0.1% Triton ~-100) prior to loading. Proteins were eluted at a flow rate
of
1 ml/min with a Linear gradient of 0-0.~ M NaCI in buffer B over 20 fractions
( 1 ml
fraction volume), followed by a second gradient of 0.~-1.0 M NaCl in buffer B
over ~
fractions. Phosphotyrosine-containing proteins were detected within fractions
7-21
according to anti-phosphotyrosine immunoblotting. The same procedures were
followed for PTP1B, with the eYCeption that the cells were not treated with
pervanadate.
For dephosphorylation reactions. Iysates of pervanadate-treated HeLa
cells ( I -? mg protein/ml) containing tyrosine phosphorylated proteins were
incubated
on ice in the absence or presence of purified active PTPs at a concentration
of ? nl~I.
Dephosphorylation was terminated by the removal of aliquots (30 ug protein)
into SDS
1~ PAGE sample buffer. and the e:~tent of dephosphorylation was determined by
itrununoblotting using the phosphotyrosine-specific monoclonal antibody G10~
generated as described below. Assays of PTP activity using tyrosine
phosphorylated
'zP-labeled reduced and carborramidomethylated and maleylated lysozyme (RC~t
lysozvme) as substrate were performed as described in Flint et al. ( 1993
E_LIBU J.
12:1937-1946).
Antibodies and ImmunoblottinQ: The PTP-PEST-specific monoclonal
antibody AG25 was raised against baculovinLS-e:cpressed purified full-length
PTP-
PEST. The anti-phosphotyrosine monoclonal antibody 6104 was generated using as
antigen phosphotyrosine. alanine and alycine. in a 1:1:1 ratio. polymerized in
the
?5 presence of keyhole limpet hemocyanin with 1-zthyl-~-(~'-
dimethvlaminopropyl)carbodiimide. a method originally described in Kamps and
Setion
(Oncogerre ?:30~-31 ~ ( 1983)). p 1 ~0~ monoclonal antibody was from
Transduction
Laboratories (Le:cington. Ky). Vlonocional antibody FG6 against PTP I B was
provided
by Dr. David Hill (Calbiochem Oncogene Research Products. Cambridge. Vt~).
Visualization of proteins by immunoblottina was achieved by enhanced
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
77
chemiluminescence (ECL) using HRP-conjugated secondary antibodies (_~mersham
Life Science Inc.. Arlington Heights. I1) and the SuperSignal"" CL-HRP
substrate
system (Pierce. Rockford. I1).
Immunoyrecipitation and Substrate Trains: Immunoprecipitation of
PTP-PEST from transfected COS cells was performed following covalent coupling
of
monoclonal antibody AG25 to protein A-Sepharose beads (Pharmacia Biotech Inc.,
Uppsala, Sweden) using the chemical cross-linking agent dimethyl pimelimidate
(Schneider et al., J. Biol. Chem. ?~ % :10766-10769 (1982)). Antibody was
first bound to
protein A-Sepharose at a concentration of 1 mg/ml bead volume. and unbound
material
was then removed by three washes with 0.? M sodium borate, pH 9. Covalent
coupling
was achieved by incubation at room temperature for 30 minutes in the presence
of ?0
mVI dimethyl pimeli.midate in 0.? VI sodium borate. pH 9. The beads were then
incubated for 1 hour with an excess of 0.? YI ethanolamine, pH 3, to block any
unreacted cross-linker. and washed three times with PBS prior to storage at
~°C. Ten q1
of AG25 beads were used to precipitate transfected PTP-PEST from lysates
containing
approximately 0.375 mg protein.
Substrate trapping was performed using various PTP affinity matrices.
The full-length PTP-PEST matrix utilized covalent coupled AG25-protein A-
Sepharose
beads to which purified baculovims-expressed PTf-PEST protein was bound.
Aliquots
(10 p.1) of AG25 beads were incubated for ? hours at -~°C in 100 q1
buffer A in the
presence of 5 p.g of purified PTP-PEST (wild type or mutant forms); unbound
PTP-
PEST was then removed by washing three times 'nnth 1 ml buffer A_ The
resultant PTP-
PES'L AG25-protein A-Sepharose beads contained approximately 2 mg of PTP-PEST
per 10 ml aliquot_ Substrate trapping was also carried out with glutathione-
Sepharose
'S beads bound to bacterially-e:cpressed GST fusion proteins containing the
catalytic
domain of PTP-PEST.
PTP 1 B was also used in substrate trapping e:cperiments. In this case. the
monoclonal antibody FG6 was precoupled to protein A-Sepharose in the absence
of
c: oss-linker (~' ug antibodvi 10 u1 beads). then purified PTP t B proteins
were added in
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
~8
excess and incubated at ~°C for ? hours. Following removal of unbound
PTP 1 B, 10 u1
beads contained approximately ? ug PTP1B.
Pervanadate-treated cell lysates. or column fractions. were used as a
source of phosphotyrosine-containing proteins for substrate trapping
e:cpenments. In
general. lysates containing 0.25-0. mg protein in 0.~ ml buffer ~ (including ~
mlV1
iodoaceric acid, 10 mIVI DTT) were incubated at ~°C for 2 hours in the
presence of 10
~1 of a~nitY matrix containing approxunazeiy 2 ug of the appropriate PTP
protein.
Unbound proteins were then removed from the samples by washing three rimes
with 1
ml buffer :~, and bound material was collected by addition of ~0 ~1 SDS-PAGE
sample
buffer followed by hearing at 95°C for ~ minutes: proteins bound to the
beads were then
analyzed by SDS-PAGE followed by immunoblotting.
In transient cotrsnsfection experiments in C OS cells. PTP 1 B
dephosphorylates p210 bcr:abl but not v-abl. When the PTP1B(D181~) mutant was
expressed as a GST fusion protein, purified and incubated with lysates of VIo7-
p210
I~ cells (which overexpress p210 bcr:abl), a complex of the mutant PTP and
p210 bcr:abl
was isolated. In conk ~S~e phosphorylated c-abl. which was also present in the
lysates. did not bind to the mutant PTP. The interaction between PTP1B(D131A)
and
p210 bcr:abl was blocked by vanadate. suggesting that the interaction involved
the
active site of the PTP.
Following transient coe:cpression in COS cells. PTP1B(D131A) formed
a comple:c with p210 bcr:abl. The YI77F mutant form of p210 bcr:abl did not
interact
with PTP1B(D181~), suggesting that this tvrosme residue is a component of the
b~~g ~m ~ the PTK. 'Ibis tyrosine residue in p210 bcr:abl is phosphorylated in
vivo
and has been demonstrated to serve as a docking site for GR.B2 (Pende~ast et
al.. 1993
~5 Cell 75:175). Direct interaction of the pTyr in p210 bcr:abl and the SH2
domain of
GR.B2 is essential for the transforming actl~ry of the PTI~ Interaction of
PTP 1 B(D 131:0 with p=10 bcr:abl interferes with the association of the PT's
~"i~
GRB=. Taken together. these data suggest that p? 10 bcr:abl is a physiological
substrate
of PTP 1 B and that PTP 1 B may function as an antagonist of the oncoprotein
PTK in
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
~9
vivo. The Vma.Y, Km and Kcat of 37 kDa PTP1B mutants toward RCV1L are shown in
Figure ?.
PTP 1 B and the EGF Receptor. Expression of PTP 1 B(D 181 A) in COS
cells leads to enhanced phosphorylation of tyrosyl residues in a 180 kDa
protein and in
proteins of 1?0 and 70 kDa. When a GST-PTP1B(D181A) fusion protein is
e:cpressed
in COS cells and precipitated on ~utathione-SepharoseT'~', the 180 kDa, and
smaller
quantities of pl?0 and p70, were coprecipitated. The p180 protein was
identified as the
epidermal ~owth factor (EGF) receptor by immunoblotting. The identity of the p
120
gad p70 proteins is unclear, however, the latter is not src, p62 or paxillin.
E.Ypression of PTP1B(D181A) in COS cells induces tyrosine
phosphorylation of the EGF receptor in the absence of its ligand. EGF,
indicating that
the mutant PTP is eYertins its efFects in the intact cell and not post-lysis.
The
equivalent PTP-PEST(D 199A) mutant_ which has the corresponding aspartate at
position 199 replaced with alanine, does not interact with the EGF receptor,
indicating
the specificity of this substrate interaction.
Autophosphorylation of the EGF receptor is required for the interaction
with PTP 1 B(D 181 A). Mutants of the receptor that are either kinase-dead or
in which
the autophosphorylation sites have been deleted do not interact with PTP 1 B(D
181 A).
In v-src-eapressin~ cells. a plethora of tyrosine phosphorylated proteins were
observed.
but phosphoryiation of the EGF receptor was not detected. Under these
conditions,
PTP 1 B D 181 A bound predominantly to a 70 kDa tyrosine phosphorylated
protein.
PTP 1 B thus appears capable of modulating EGF-induced signaling pathways.
E~.-WtPLE ~
PTP-PEST PREFER~NThLLY DEPHOSPHORYL.aTES ~1 1~0 KD.4 PHOSPHOTYROSINE
7j CO'iT.~lN1'iG PROTEIN
In order to investisate the substrate specificity of PTP-PEST in vitro.
aliquots of pervanadate-treated HeLa cell lvsates were incubated on ice.
yielding ~0-100
distinct phosphotyrosine-~:ontaining proteins as judged by immunoblottina of
the cell
CA 02375145 2001-11-23
WO 00/75339 6o PCT/US00/14211
lysate using the monoclonal anti-phosphotyrosine antibody G10~. Purified full-
len'th
PTP-PEST (expressed in SP9 cells using recombinant baculovirus), PTP-PEST
catalytic
domaitL or PTP1B catalytic domain (~~ kDa form) was then added to the lysate.
and
aliquots were removed at various time points for analysis by SDS-PAGE followed
by
anti-phosphotyrosine immunoblotting.
Surprisingly, a prominent 130 kDa phosphotyrosine band (p130) was
selectively dephosphorylated by PTP-PEST within 10 minutes, whereas the
intensity of
all the other bands was essentially unchanged even after 60 minutes of
incubation with
PTP-PEST. Long incubations with higher concentrations of PTP-PEST (eater than
I00-fold) resulted in the complete removal of alI phosphotyrosine bands from
the
lysate. However, under all conditions tested, pl.i0 was found to be
dephosphorylated
more rapidly than all other bands present.
The selective dephosphorylation of p130 by PTP-PEST was also
observed using a truncated form of the phosphatase (amino acid residues 1-30~)
which
I S essentially contains only the catalytic domain of the enzyme. This result
suggests that
the striking substrate preference displayed by PTP-PEST in this analysis is an
inherent
propemr of the phosphatase catalytic domain, whereas the C-terminal X00 amino
acid
residues have little discernible effect on the substrate specificity of the
enzyme.
The specificity of the interaction between PTP-PEST and p 130 was
examined using the catalytic domain of PTP 1 B (amino acid residues 1-X21 ) in
dephosphorylation reactions. When added at a similar molar concentration to
that used
for PTP-PEST. PTP1B was found to dephosphorylate fully and rapidly (within 1~
minutes) most of the phosphot~rrosine-containing proteins present in the
pervanadate
treated HeLa lysate. In addition- the time course of dephosphorylation of p 1
~0 was not
2~ significantiv_ more rapid that. that of the other phosphotyrosine bands
dephosphorylated
by PTP 1 B . The range o f PTP 1 B substrate specificity in vitro and in vivo
thus can differ
where availability of a Given substrate may vary. and where an isolated PTP
catalytic
subunit is characterized_
CA 02375145 2001-11-23
WO 00/75339 6I PCT/US00/14211
EY~vIPLE ~
IDEVTIFIC.aTION OF A I~O KDA SL.BSTR.1TE OF PTP-PEST BY SUBSTR.~TE TRAPPING
This e:cample describes the use of a substrate trapping mutant PTP in an
affinity matti.Y. to identify a PTP substrate in a cell lysate. For
preparation of the
substrate trapping PTP at~nity mama, a mutant form of PTP-PEST (D 199A) was
generated by site-directed mutagenesis. and the mutant enzyme was purified
following
e:~pression using recombinant baculovirus. When assayed using tyrosine
phosphorylated RC1~I-Lysozyme as substrate. the purified mutant enzyme
exhibited a
specific activity which was approxunately 10.000 fold lower than that of the
wild type
enzyme. This purified protein was bound to an afEnity matri:c comprised of an
anti-
PTP-PEST monoclonal antibody (.-~G25) covalently coupled to Protein :~-
Sepharose
beads. then incubated with each of the Vlono Q fractions prepared from HeLa
cell
lvsates as described in E~ataple 3.
Pervanadate-treated Hei.a cell lysate was fractionated by anion e:cchange
chromatography (E.~cample 3) and aliquots of the fractions were analyzed by
SDS
PAGE followed by immunoblotting with anti-phosphotvrosine or anti-p130'~
antibodies. Aliquou of all samples analyzed were then incubated with an
affinity
matrix containing a subswate trapping PTP-P~ST mutant. comprising full length
PTP
PEST in which Asp199 is changed to alanine (D199A), bound to covalently
coupled
protein A-Sepharoserantibody (AG2~) beads. After ~5 minutes of incubation.
proteins
associating with the mutant PTP-PEST were collected by centrifugation. the
beads were
washed, and SDS-PAGE sample buffer was added. Associated proteins were then
analyzed by itnmunoblotting using the monoclonal anti-phosphotyrosine antibody
G10~. Proteins associated with PTP-PEST were then analyzed by SDS-P.aGE
followed
~5 by immunoblotting with anti-phosphotyrosine or anti-pl.i0'~ antibodies.
anti-phosphotvrosine immunoblotting of the column fractions showed
that the p 1 ~ 0 phosphoy rosine band eluted as a single peak in fractions 11-
1 ~ (approx.
0.~ VI V aC 1 ). In view of the abundance of tyrosine phosphorylated p 1 ~ 0
in HeLa
lysates. it appeared likely that p 1 ~ 0 represents a previously identified
phosphotyrosine-
CA 02375145 2001-11-23
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6'
containing 130 kDa protein. Several potential candidates were identified in
the
literature. including the focal adhesion kinase p 1?5F''~". ras-GtIP. op 130
and p 130'. Of
these candidates. p 130' has been identified as a particularly prominent
phosphotyrosine band in a wide variety of systems, including v-crk (~Iayer and
Hanafusa. Proc. ~Vatl. .cad Sci. LSd 8::?638-2642 (1990); Vlayer et al.,
:Vature
332:272-275 (1988) and src (Kanner et al., Proc. Natl. cad Sci. G'S~ 87:3328-
3352
(1990); Reynolds et al., nlol. Cell. Biol. 9-.3951-3958 (1989)) transformed
fibroblasts,
integrin-mediated cell adhesion (Nojitna et al.. J. Biol. Chem. 270:15398-1502
(I995);
Perch et al.; .I. Cell Science 108:1371-1379 ( 1995); Vuori and Ruoslahti, J.
BioL Chem.
Z~O:~~~ 9-?262 (1995)) and PDGF stimulated 3'I ~ cells (Rankin and Rozengurt,
J.
Biol. Chem. 269:704-7I0 (1994)).
Therefore. the possibility that the p 130 phosphotyrosine band
corresponds to p130'y was tested by immunoblotting the Vlono Q fractions using
an
antibody to p130'~. The 130 kDa band corresponding to p130'u eluted in the
same
fractions as the p 130 tyrosine phosphorylated band, and displayed a similar
apparent
molecular weight, suggesting that they might represent the same protein.
Furthermore,
p130'~ immunoprecipitated from these fractions was found to be phosphorylated
on
tyrosyl residues.
The mutant PTP-PEST protein was found to associate with a single
phosphotyrosine-containing protein. the molecular weight (I30 kDa) and Mono Q
elution position (fractions 11-1=~) of which coincided with those of p 130.
Immunoblotting of the PTP-PEST-associated proteins using the p130~ antibody
demonstrated that the 130 kDa tyrosine phosphorylated protein trapped. by the
mutant
PTP-PEST is indeed p130'u. Therefore it appears that p130"~ is a
physiologically
relevant substrate for PTP-PEST.
Structural Features of PTP-PEST in S ecinc Interaction with Tyrosine
Phosphorv lated o 130'w The interaction between P 130'' and PTP-PEST was
investisated further in substrate trapping experiments using various purified
mutant
forms of PTP-PEST to precipitate proteins from pervanadate-treated HeLa
Lvsates.
Several ai~nity matrices were incubated with pervanadate-treated HeLa cell
tysaie. and
CA 02375145 2001-11-23
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6~
proteins associated with the beads were analyzed by SDS-PAGE followed by
itnmunoblotting with anti-phosphotyrosine or anti-p130"' antibodies.
The wild type full-length phosphatase was found to be incapable of
stable association with tyrosine phosphorylated p130"~, whereas both the PTP-
PEST
(D 199A) mutant protein and a mutant lacking the active site cysteine residue
(C231 S)
specifically precipitated p130'~ from the lysate. The inability of the wild
type
phosphatase to precipitate tyrosine phosphorylated p130"~ presumably reflects
the
transient nature of the normal interaction between PTP-PEST and tyrosine
phosphorylated p1~0~, which is likely to be concluded as soon as p130"~ is
dephosphorylated by PTP-PEST.
Since the C-terminal X00 amino acids of PTP-PEST contain several
proline-rich regions which resemble src homology-~ (SH3) domain binding
sequences.
it appeared plausible that the specificity of the interaction between PTP-PEST
and
p130'~ might depend to some extent on association of these segments with the
SHS
1 ~ domain of p 130". The possible contribution of the C-terminal se~nent of
PTP-PEST
in the observed specific interaction of PTP-PEST with p130~ was therefore
addressed
in further substrate trapping experiments using GST fusion proteins containing
the
catalytic domain of PTP-PEST alone, in both wild type and mutant (D199A)
forms.
The mutant catalytic domain of PTP-PEST fused to GST was found to precipitate
the
p130"~ phosphotyrosine band specifically, whereas both the wild type fusion
protein
and GST alone failed to precipitate p130'~. The specific interaction between
PTP-
PEST and p130"~ observed in these experiments therefore appears to be an
intrinsic
propem of the catalytic domain of PTP-PEST, emulating the observed preference
of
the active PTP-PEST catalytic domain for dephosphorylation of p130°s in
vitro.
S ecificitv of Interaction Between Mutant PTP-PEST and Tyrosine
Phosnhorvlated t?l30'w In view of the relative abundance of tyrosine
phosphorylated
p l ~ 0'~ in the pervanadate-treated HeLa cell Ivsate. the possibility that
the observed
selective binding of PTP-PEST inactive mutant proteins to pI.O''~ was
substrate-
directed (;retlecrin~ the abundance of this potential substrate relative to
the other
phosphotyrosine-containing proteins present in the lvsate) rather than enzyme-
directed
CA 02375145 2001-11-23
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6~
(reflecting a genuine substrate preference of PTP-PEST) was considered; this
possibility
was addressed in two ways. First inactive mutant fotTns of the catalytic
domain of
PTP I B were used to trap potential substrates for this enzyme from the
pervanadate-
treated HeLa lysates. Again it was found that the wild type phosphatase was
incapable
of stable interaction with any phosphotyrosine-containing protein. whereas
mutant
variants of the PTP1B phosphatase domain (comprising Cys or Asp mutations
analogous to those described above for PTP-PEST) associated with many tyrosine
phosphorylated proteins. This was especially apparent for the aspartic acid
mutant of
PTP 1 B (D 181 A), which appeared to precipitate essentially all
phosphotyrosine-
IO containing proteins from the lysate with similar efficacy. These data
emphasize the
specific nature of the interaction between PTP-PEST and p 1 ~ 0~. which
appears to be a
propem peculiar to the PTP-PEST catalytic domain. rather than a feature shared
by all
PTP catalytic domains.
The specificity of the interaction between PTP-PEST and p 130'u was
1~ addressed further following pervanadate-zr~azment of several different cell
lines (Wi38,
293, COs. MCF10A, C2C12. MvLu), yielding a different array of tyrosine
phosphorylated proteins in each case: the resultant lysates were analyzed by
SDS
PAGE followed by anti-phosphotyrosine itnmunoblotting. Aliquots were incubated
with PTP-PEST (D 199A) a~niry matri:c or control matri.~c. and tyrosine
phosphorylated
20 proteins associating with PTP-PEST were analyzed by SDS-PAGE and
immunoblotting
with anti-phosphotyrosine or anti-p130''~ antibodies as described above.
In each case. the D 199A mutant PTP-PEST protein precipitated a single
broad phosphotyrosine band with an apparent molecular weight between 1?0 and
150
kDa in different cell lines. whereas the affinity matri.Y alone failed to
precipitate any
~5 phosphotyrosine-containing protein. Immunoblottins of the precipitates with
a p130"5
antibody revealed that the protein precipitated from all cell lysates
corresponded to
p 130"S; the observed molecular weight variation between different cell Lines
presumably retlec~~s either species differences in the molecular weight of
p130"s or
e:cpression of different alternatively spliced forms (Sakai et al.. E LIBO J.
I ~ :37-~8-:'~ d
30 ( 1990).
CA 02375145 2001-11-23
WO 00/75339 6 PCT/US00/14211
The relative abundance of tyrosine phosphorylated p 1 ~ 0'~ in the PTP-
PEST precipitates appeared to correlate approximately with the abundance of
p130'~
protein in the lvsates (data not shown). Surprisingly. re5~~ess of the
abundance of
tyrosine phosphorylated p130"~ in the lysates. p130''~ was invariably the only
phosphoryrosine-containing protein in the precipitates. even in ?93 cell
lysates which
contained very little p I ~ 0'u protein but which displayed a wide variety of
other
abundantly tyrosine phosphoryiated proteins. Similarly, when lysates of
pervanadate-
treated 293 cells (containing tyrosine phosphoryiated p130'~ in amounts which
are
undetectable by anti-phosphoryrosine immunobiotting of the lysate) were
incubated
with active PTP-PEST, no visible dephosphorylation of any phosphotyrosine band
occurred (Garton and Tonks, unpublished data). These results indicate that the
affinity
of PTP-PEST for p130"~ is substantially 'eater than for any other substrate
present
and further emphasizes the remarkable substrate selectivity of PTP-PEST for p
130'.
Vanadate Inhibition of Tyrosine Pho horvlated 130' :association with
1 ~ Mutant PTP-PEST: A consistent observation was that in contrast to the
inactive
mutant PTP-PEST, the wild type enzyme failed to associate in a stable comple:c
with
tyrosine phosphorylated p 130', suggesting that the observed association is
active site
directed. In order to investigate this possibility. mutant PTP-PEST (D 199A)
was
incubated with the PTP inhibitor vanadate (Denu et al., 1996 Proc.. Natl. .cad
Sci GSA
93:?493-?498), at various concentrations prior to addition of pervanadate-
treated HeLa
cell lysate. The e:ctent of association of p130~ with PTP-PEST was then
analyzed.
PTP-PEST affinity matri.~c. comprising full length PTP-PEST (D 199A) bound to
covaleatly coupled protein A-Sepharose~antibody (AG25) beads, was incubated
for 10
minutes on ice in the presence of varying concentrations of sodium
orthovanadate. The
''_5 samples were then incubated with aliquots of pervanadate-treated HeLa
cell lysate:
associated proteins were analyzed by SDS-P AGE and immunoblotting with ann-
phosphoryrosine or anti-p 130' antibodies. The activity of wild type PTP-PEST
was
also determined ;order the same conditions. using tyrosine phosphoryiated '-P-
labelled
RCS(-lvsozyme as substrate.
CA 02375145 2001-11-23
WO 00/75339 66 PCT/US00/14211
The association was found to be potently disrupted by vanadate. with a
concentration-dependence similar to that of vanadate inhibition of wild type
PTP-PEST.
and complete disruption being observed at 10 tniVt vanadate.
E~,;~tPLE 6
ASSOCIATION OF EVDOGF'10US P1~0''~ 'KITH TR~.NSFECTED MLT.~NT PTP-PEST IN
COS CELLS
The work described above strongly suggests that p 130"s represents a
physiological substrate of PTP-PEST. In order to assess whether PTP-PEST
interacts
with p130'~ in intact cells. COS cells were transfected with plasmids encoding
wild
type or substrate trapping mutant forms (D 199A or C?31 S) of PTP-PEST. The
cells
were treated with pervanadate 30 minutes prior to lysis. PTP-PEST proteins
were
immunoprecipitated. and associated tyrosine phosphorylated proteins were
analyzed by
anti-phosphotyrosine immunoblotting of the resultant precipitates. Lysates
were also
incubated with covalently coupled protein A-Sepharoselanti-PTP-PEST (AG25)
beads
and associated proteins were analyzed by SDS-PAGE and immunoblotting with anti-
phosphotyrosine antibody.
Under these conditions. the phosphotyrosine-containing band
corresponding to p 1.i0'~ was again unique in its ability to associate with
the CZ~ 1 S
PTP-PEST protein. indicating that p 130"s can be specifically selected by PTP-
PEST as
a substrate in an intracellular conte.~ct in the presence of a lame number of
alternative
possible subsnates. Neither the wild type nor the D199A form of PTP-PEST was
capable of a stable interaction with tyrosine phosphorylated p 130" in
pervanadate-
treated COS cells.
The binding of both wild type and D 199 PTP-PEST to tyrosine
?5 phosphorylated p1~0'~ under these conditions is most likely prohibited by
the presence
of pervanadate bound to the active site cysteine residue of PTP-PEST (Denu et
:11..
Proc. Varl. .-lcad. Se;:. LSD 93:'~9~-?~93 ( 1996)). which effectively
e:ccludes the
binding of phosphotyrosine residues of p 130'. The ability of the C'_'~ 1 S
mutant PTP
CA 02375145 2001-11-23
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67
PEST to associate in a stable complex with p130"~ in the presence of
pervanadate
suggests that this mutant protein is largely unaffected by pervanadate,
indicating that
the normal mode of inhibition of PTPs by vanadate ions depends critically on
direct
interactions between vanadate and the thiolate anion of the PTP active-site
cysteine
residue. These observations therefore lend further support to the existence of
an
exclusive interaction between PTP-PEST and p130~. which appears exclusively to
involve the PTP-PEST active site, and therefore reflects the physiological.
highly
restricted substrate preference of PTP-PEST for p130°'.
E~~1~LE 7
~~ PREPAR.aTION OF SLBSTR:.TE TR:IPQiVG PTP MZ;TaNTS
Generation of mutant PTPs capable of interacting with substrates in a
stable complex was essentially as described (Flint et al.. 1997 Proc. Vat. .-
lcad Sci.
USA 9:1680; Garton et al., 1996 :Llol. Cell Biol. 16:608: Tiganis et al., 199?
J. Biol.
Chem. Zi2:215~8; see also PCT US97/13016). Plasmid isolation. production of
1~ competent cells, transformation and M13 manipulations were carried out
according to
published procedures (Sambrook et al., .ylolecular Cloning, a Laboratorv
:Llam,~al, Cold
Spring Harbor Laboratory Press. Cold Spring Harbor, NY, 1989). Purification of
DN
fragments was achieved using a QIAEX~' kit purchased from QI~GEN. Inc.
(Chatsworth. Cue). Sequencing of the different constructs was performed using
a
?0 SequenaseT'~' kit (~mersham-Pharmacia. Piscaraway, N~ according to the
manufacturer's instructions. Restriction and modification enzymes were
purchased
from Roche Molecular Biochemicals (Indianapolis. IN) and New England Biolabs
(Beverly. l~fr1).
Brietlv. human PTPH1 cDNA (U.S. Patent No. ~.~95.911) liaated into
?s plasmid pBlueScript (Stratagene. LaJolla. Ca) was mutated by site-directed
mutagenesis using the Viuta-GeneT'~' kit (Bio-Rad. Inc.. Hercules. C.~)
according to the
supplier's instructions. T'ne oli~onucleotide used for in vitro mutaUenesis of
cysteine
8-~~ to serine was:
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
68
CCT SGT TC ~ CTC CMG TGC TGG :~1T ~.G SEQ ID N0:37
which spans nucleotides ?~~7-?562 of PTPH1. The oligonucleotide for
mutagenesis of asparcate 311 to alanine was:
GCA TGG CCT GCC CAC GGT GTG C SEQ ID N0:~8
which spans nucleotides 2~~-?~6 of PTPH1. The mutated replicative
form DNr1 was transformed into E coli strain DH10B (Stratagene, La Jolla. CA)
and
colonies were picked and dideoxy sequenced using a SequenaseT'~' kit (Amersham-
Pharmacia. Piscataway> NJ) according to the manufactures s instructions for
verification
of the mutation. The portions of the wildtype and mutated PTPH1 ?enes encoding
the
PTP catalytic domain (amino acid residues 63~ to 913) were ligated in-frame
into the
1 ~ e:cpression vector pGE.~ (~mersham-Pharmac~a, Piscataway, Nn to generate
three
glutathione-S-transferase (GST) fusion protein encoding sequences: GST-
PTPH1(w-ildtype), GST-PTPHl(D811:~) and GST-PTPH1(C842S). GST-PTPH1
fusion proteins were e:cpressed in E. coli and purified by affinity binding to
?lutathione
immobilized on Sepharose'''~' beads (Pharmacia_ Piscatawa; NJ) according to
the
manufacturer s protocol.
:alternatively. wildtvpe and mutant PTPH1 constructs as described above
to be used for transfection of mammalian cells were tagged at the C-terminal
encoding
ends with nucleic acid sequences encoding the HA epitope_ The HA tag
corresponds to
an antibody -de:ined epitope derived from the influenza hemaaglutinin protein
(~Nilson
.5 et a1._ 198- Call 3; :767):
SYPYDVPDY:~S SEQ ID N0:39
after confirmation by DNA sequencins, these constructs we:e cloned
into vector pCDN.~~ (InvitroQen. Carlsbad_ C.~) and retroviral vector pBSTRI
~S.
Reeves. Massachusetts General Hospital. Boston. VLF).
CA 02375145 2001-11-23
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69
PTPH 1 (D811 ~) mutant constructs were further modified by size directed
mutagenesis as described above but using the oligonucleotide:
TTG GAC ~ ~1C CGA TTT :~ GAT GTG CTG CCT TAT G SEQ ID NO::~O
which spans nucleotides 2034-2070 of PTPH1 to ?enerate a double
mutant (Y676F1D811A) in which the conserved PTP catalytic site tyrosine
residue at
position 676 is replaced with phenvlalanine.
P~WIPLE 8
INFLUENCE OF PTPHI EXPRESSION ON CELL GROWTH IN TR~NSFECTED CELLS
IO This example shows that overeapression of a transfected PTPH1 gene in
cultured cells markedly impairs cell ~owth. while overe:cpression of a
transfected
mutant substrate-trapping PTPHI gene does not.
Stable ~2T3 cell lines e;cpressing wildtype or substrate trapping
mutant PTPH1 GST fusion proteins (see E:cample 7) under the control of a
tetracycline
1 ~ repressible promoter were constructed using a retroviral gene delivery
system (Paulus et
al.. 1996 ,I. virol. l 0:62; Wang et al.. 1998 Genes Develop. I ?: I769).
Briefly.
confluent 10 cm diameter tissue culture plates of the viral packaging cell
line Lin:~ (G.
Hannon_ Cold Spring Harbor Laboratory, Cold Spring Harbor. NY) were
transfected by
calcium-phosphate precipitation with 1~ ~ of either the wildtype or mutant
D811A
20 PTPHI retroviral constructs. To maintain repression of PTPHI gene
e:cpression. the
following steps of establishing and maintaining the stable cell Iines were
performed in
the presence of 2 uQlml tetracvciine (Clontech. Palo :alto. C:~). Retroviruses
were
produced by culturing the transfected Lin.~ cells at 30°C for ~3 hours
afrer which
culture fluids containing virus were filtered using a 0.~.~ um filter
(vlillipore. Bedford.
''_S VIA) to remove packaging cells. The viral supernatants were supplemented
with -~
uQiml polvbrene (Sigma. St_ Louis. VIO) and were used to infect ~iIH3T: cells
(Cold
Spring Harbor Laboratory stock. orisinally obtained from :American Type
Culture
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
Collection. Rockville, vfD) maintained in Dulbecco's modified Eagle's Vfedium
(DIvIEVt, GIBCO-BRL, Grand Island. N~ supplemented with 10% fetal bovine setwn
(FBS, GIBCO-BRL). Infection took place overnight at 30°C, after which
the medium
was replaced with fresh medium and cultures were incubated at 37°C. Two
days later
selective conditions were imposed by supplementing the medium with puromycin
to a
final concentration of 2 ugiml. Individual colonies were isolated and
maintained in the
presence of both tetracycline and puromycin. To induce PTPHI expression, cells
were
washed and re-seeded in new dishes in the presence of puromycin. but in the
absence of
tetracycline.
10 Cell ~owth was markedly ~bited (approximately seven-fold decrease
in accumulated cell number) when wildtype PTPH1 catalytic domain expression
was
induced by removal of tetracycline from the culture media (Figures ~ and -f).
Approximately 10% of the cells ~adually detached from the culture dish during
induction of wildtype PTPH1 e:cpression_ and these cells were non-viable as
determined
1 ~ by their inability to exclude trypan blue. In contrast expression of the
catalytically
impaired PTPHI-D81 IA mutant ("DA~7 had no effect on cell ~owth or viability.
For
each PTPH1 construct, similar results have been obtained in three separate
cell lines
generated from distinct isolated colonies. indicating that differences among
clonal
populations do not account for the phenotypic differences observed between
cells
20 transfected with wildtvpe and mutant (D811?.) PTPH1. Using a DNA
fra~entanon
assay (Wyllie. 1980 :Varz~re ?8-I:5>j: Arends et al.. 1990 Vim. ,I. Parhol.
136:~9~), it was
determined that cells in which PTPH1 expression was induced did not undergo
apoptosis.
Cell cycle analysis by flow cvtotluorimetric measurement of DNA
~5 content using propidium iodide (Rabinovitch. 199 _~leths. Cell Biol. ~1:?63-
?96) was
performed on populations of transfected cells in which wiidtype or mutant
(D811~)
PTPH1 e:cpression was induced. The distribution of cells amonQSt various
phases of the
cell cycle was not altered relative to control cells. indicating that PTPH1-
induced
growth arrest did not operate in a particular cell cycle phase.
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
7I
Cells in culture were also synchronized to determine the effects of
PTPH1 expression on re-entry into the cell cycle during recovery from Gl/S
arrest.
Following a ?~-hour period of induced PTPHl (wildtype or mutant D811A)
expression,
cells were 5-vnchronized by cultivation for 18 h in the presence of 1 tnVl
hydroxvurea
(Calbiochem, San Diego. C A) : this agent arrests cells at the G1/S boundary
in the cell
cycle (Kreck and DeCaprio, 1995 .Lleths. En.:-ymol. ?5~:11~) . The hydroxyurea
block
was released by washing the cells with flesh medium three yes. At various tune
points following removal of the cell cycle block. cells were lysed in NP40
buffer (1%
NP40. 10 nuVl sodium phosphate-pH 7.0, 1~0 tnVt NaCl. ? tnul EDTA, ~0 tnul
NaF, 1
mVI Na~VO~, ~ ~g/ml leupeptin, ~ ~glm1 aprotuiin. 1 mui benzamidine, 1 muI
P'~ISF)
for immunoblot analysis using a cyciin-specific antibody. Briefly, confluent
cells in a
10 cm diameter tissue culture plate were lysed at -~°C for I O min in
0.~ ml ~iP~O buffer.
and the lysates were clarified by centrifugation at 10.000 Y g for 10 min at
=~°C.
Aliquots of each lysate were normalized for protein concentration (BCA assay.
Pierce
1~ Chemicals, Rockford_ IL), diluted in sodium dodecylsulfate (SDS) sample
buffer
(Laemmli. 1970 Nature 227:680), resolved by SDS poiyacrylamide gel
electrophoresis
using 8% acrylamide gels and blot transferred onto Immobilon-P PVDF membranes
(Llillipore, Bedford. VIA). Polvcional rabbit anti-cvclin D 1 antibodies
(Santa Cruz
Biotechnology. Santa Cruz. CA) diluted according to the supplier's
recommendations in
immunoblot buffer ('_'0 mVI Tris-pH 7.~ containing ~% (wiv) nonfat dry milk.
1~0 muI
NaCI and 0.0~% Tween ?0) were used to probe the blot for 1 hour at room
tempezature.
The blot was washed three times in the same buffer and developed using
enhanced
chemiluminescence (ECL) reagents and horseradish peroxidase (HRP)-coupled
secondary antibodies (both from Amersham-Pharmacia Biotech. Piscataway, New
''S Jersey) according to the supplier s instructions. as previously described
(Zhang et al..
199 J. Biol. Chem. ?-0:'_'006.
As shown in Figure ~. when transfected cells were released from the
hydroxvurea cell cycle block under conditions non-permissive for expression or
the
w-ildtvpe PTPH1 transVene. cvclin D expression gradually increased as cells
reentered
~0 and progressed through the cell cycle. When. however. cells were released
from the cell
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
7?
cycle block under conditions permissive for PTPHl eYpression_ all detectable
cyciin D
expression was abolished. suggesting that PTPH1 retards cell ~owth by
disrupting cell
cycle progression. E.~cpression of a mutant PTPH1(D811~) in cells transfected
with the
mutant transgene had no effect on the cell cycle.
E~C.:~vIPLE 9
IDE~ITIFIC.3TION OF VCP ~s ..~ PTPH1 SussTx~,TE UsmrG .~ PTPH1 SussT~TE
'fR;,ppn~tG ViUT~NT IN VITRO
'This e:cample describes identification of a PTPH1 substrate in cell
lysates, using a substrate trapping PTPH1 mutant having the invariant PTP
catalytic site
aspartate residue replaced with alanine (D811 ~). Cell lysates were prepared
as
described above in E.~campie 3, and then contacted with wildtype or mutant
PTPH1
catalytic domains to determine PTP-substrate binding interactions.
Substrate trapping methodologies using mutant PTPs in which the
invariant catialytic domain asparate residue is replaced with alanine were as
described
1~ (Flint et al.. 1997 Proc. Vat. .-lcad Sci. USA 9:x:1680; Garton et al..
1996 :Llol. Cell
Biol: 16:608: Tiganis et al., 1997 J. Biol. Chem. ?; ?:21 ~~8; see also PCT
US97/13016) except that the mutant PTP was PTPH1 (D811A) as described above in
Example 8.
Pervanadate-treated cell lysates were incubated with GST-PTPHI
catalytic domain fusion proteins immobilized on SepharoseT'2 beads. Briefly,
subconfluent mammalian cell cultures were treated with ~0 E.Wf pervanadate
(diluted
from a 1:1 mi.~cture of 100 rm~i sodium vanadate and 100 rW( H,0= in DhfEW)
for ~0
min_ washed with PBS and lysed_ as described in E.Yample 8. in substrate-
trapping
buffer (1°,'o Triton Y-100. ~0 mVI HEPES-pH 7.~. ~ mV( EDT:. 1~0 rruW
NaCI. 10 mNl
via phosphate_ ~0 mW VaF. ~ mu( iodoacetic acid_ ~ uJml leupeptin. ~ uglml
aprotinin. 1 mW benzamidine and 1 WI PNISF. Lysates were made l0 cnVl DTT and
ciari~ied by centrifu=anon for 10 min at 10.000 ~c g. Purified GST-PTPH1
fusion
proteins. or GST alone. bound to ~lutathione-Sepharose beads ( ~mersham-
Pharmacia
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
7~
Biotech, Piscataway. NJ) under conditions recommended by the supplier were
e:ctensivelv washed with phosphate buffered saline (PBS) containing 1°%
Triton X-100
(Si~na- St. Louis. V10), ? ttWl diththiothreitol (DTT. Sigma), ~ uglml
leupeptin, ~
~g/ml aprotinin, 1 cniVl benzamidine and 1 tnlVl PI~ISF. Lysates were
incubated with the
bead-immobilized GST or GST-PTPH1 catalytic domain fusion proteins for 2 h at -
~°C,
and the beads were washed four times with substrate-trapping buffer. Material
bound to
the beads was resolved by SDS-PAGE and blotted onto Immobilon-PT'~'
(l~fillipore,
Bedford, MA) membranes, then probed with phosphotyrosine-specific monoclonal
antibodies at concentrations recommended by the suppplier (G98. Tiganis et
al.. 1997 J.
Biol. Chem. l l 2:21 X48; =1G10, Upstate Biotechnology. Lake Placid, NY;
P'j?0.
Transduction Laboratories. Lexington, K'~ and developed using ECL reagents
(A.mersham-Pharmacia Biotech, Piscataway, NJ) as described above in E.Yample
7.
~, prominent, tyrosine-phosphorylated protein of 97 kDa (pp97) was
specifically isolated by the PTPH1(D811~-1) mutant from 293 cell lysates. but
not by
1~ either the wildtype PTPH1 or the PTPH1(C8~2S) mutant (Figure 6).
Furthermore,
pp97 was also consistently recovered by PTPH1(D811A) as the major tyrosine-
phosphorylated protein from other mammalian cell lines tested. including A431,
COS-
7, HepG2, MDCK. REF-~2. Saps-2 and Vero cells. The PTPH1 substrate trapping
mutant specifically and preferentially bound to pp97, which was one of several
hundred
tyrosine-phosphorylated proteins present in the cell lysates; pp97 was not a
major
protein component in any of the cell lysates used as a starting material for
substrate
trapping. Variable amounts of other, minor tyrosine-phosphorylazed proteins
were also
detected in the PTPH1-associated materials from the various cell lines.
Purification of pp97 on immobilized PTPH1(D811r~) from lysates
representing 10g 29~ cells was scaled up to obtain sufficient protein for
partial
sequencing by Edman de~adation of K-endopeptidase-digested peptides (Russo et
31..
199'_' J. Biol Chem. .6 7 :20317). Sequences of seven individual peptides were
determined '(Figure ~l. all of which were Found to match amino acid sequences
present
in a membrane-associated protein having .~TPase activity and known as p97 or V
CP
~0 (E~erton zt al.. 1992 FLIBU f 11:333). Underlined sequences (Fig. 7)
matched the
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
74
mouse VCP sequence retrieved from the ~1CBI database
(httPV~~~ncbi.nlm.nih.jov~,
accession number Z1~044)(SEQ ID VO:=~2). The Yeast ortholog of VCP, known as
CDC~B, is a well established cell cycle revelatory protein (Patel et al., 1998
Trends Cell
Biol. 8:6~). A synthetic peptide corresponding to the C-terminal 1 ~ residues
of marine
VCP (Egerton et a1._ 199?) was prepared (Cold Spring Harbor Laboratory Core
Peptide
Facility, Cold Spring Harbor, N'~ and conjugated using SPDP (N-succinimidyl 3-
{2-
PYTidyldithio]proprionate, Pierce Chemicals, Rockford. IL) according to the
m~tt~acturer's recommendations to keyhole limpet hemocyanin (KLH, Pierce
Chemicals) for use as an immunogen according to standard procedures (Harlow
and
10. Lane, Antibodies: ~ Laboratory ~Llam~al, Cold Spring Harbor Laboratory,
1988; Weir.
D.M., Handbook of Experimental Immunology. 1986, Blacltweil Scientific.
Boston) to
generate polycional rabbit antiserum CS~31. The VCP peptide immunogen had the
sequence:
GGSVYTEDNDDDLYG SEQ ID NO:~I
1~ E~WiPLE 10
IDENTIFICATION OF VCP ~S A PTPH1 SUBSTRATE USING ~ SUBSTRATE TRAPPING
PTPHl DOUBLE MUTA'tT HAVING SUBSTITUTED ACTIVE SITE TYROSINE
'Ibis e:cample describes identification of an interaction between a PTP
and its substrate in intact cells, using a substrate trapping PTP double
mutant. More
20 specifically, this e:cample employs the PTPH1 double mutant having the
invariant PTP
catalytic site aspartate residue replaced with alanine (D811A) and also having
the
conserved PTP catalytic site tyrosine residue at position 676 is replaced with
phenylalanine.
Cultured 293 cells were transfected using the HA-tagged PTPH1
?5 constructs described in E.Yample 8. and the e:cpressed HA epitope tagged
proteins were
recovered by immunopmcipitation with HA-specific monoclonal antibody 1=C A
bound to immobilized staphylococcal protein A as described (Zhang et al.. 1997
J. Biol.
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
Chem. ~i?:?7281). Immtmoprecipitates were prepared according to stand~d
procedures from lysates produced as described above in Example
Immunoprecipitates were analyzed for phosphotyrosine-containing proteins by
western
immunoblot methods as described above. Surprisingly, the PTPH1 (D811 A) mutant
e:cpressed in 393 cells contained significant and readily detectable levels of
phosphotyrosine (Figure 8A), which contrasted with the absence of detectable
phosphotyrosine in the GST-PTPH1(D811A) fusion protein expressed in E toll
(Figure
6). From these results. the location of phosphorylated tyrosine in the PTPH1
primary
structure could not be determined. Additionally, the PTPH1(D811A) mutant
expressed
10 in 293 cells did not co-precipitate detectable pp97/VCP (Figure 8).
PTPHl(D811A)
thus failed to e~ciently trap detectable pp97/VCP in vivo in a manner
commensurate
with the in vitro pp97/VCP trapping exhibited by PTPH1(D81 1A) in vitro
(Example 9).
Analysis of the PTPH1 catalytic domain amino acid sequence revealed
the presence of a conserved tyrosine residue at position 676 in the PTP active
site
15 (Barford et al., 1995 Vat. Struct. Biol. ?:1043). An HA-tagged PTPHl double
mutant
was constructed as described in E.~camPle 8, in which the tyrosine at position
676 of
PTPH1(D811A) was replaced with phenylalanine to provide PTPHl(Y676F/D811A).
Cell lysates from ?93 cells transfected with a construct encoding the PTPH1
(Y6 7 61D811A) double mutant were lysed. immunoprecipitated with monoclonal
anti-
~p HA antibody and analyzed by western immunoblot methodologies as described
above
for the presence of phosphotyrosine. Immunoprecipitated materials were also
analyzed
for the presence of pp97/VCP using antisezum CS531 (E.oample 9), and for the
presence
of the HA epitope using monclonal antibody 12CA~ (Zhang et al., 1997 J. Biol.
Chem.
l i?:?7?81).
L mike the X93 cells transfected with the single mutant PTPH 1 (D811 A).
293 cells transfected with the double mutant PTPH1(Y676F,~D811A) had gained
the
ability to specifically trap pp97~CP- as demonstrated by immunoblot analysis
of the
itnmunoprecipate using antiserum CS>>1 (Figure 81. When analyzed for
phosphotyrosine content. the double mutant PTPHl(Y676F-D811 A)
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76
immunoprecipitated from transfected 293 cells e:chibited dramatically reduced
phosphoryrosine, relative to the single mutant PTPH1(D811A) (Figure 8B).
E~,~IPLE 11
IDE~(TIFIC~.TION OF TYROSINE PHOSPHORYL.aTION SITES ON ~ PTPH1 SUBSTR.aTE IN
VIVO USING .a SUBSTRATE TRAPPING PTPH1 DOUBLE MUT.aNT
In this e:cample, a substrate trapping PTPH1 double mutant is used to
functionally characterize tyrosine phosphoryiation sites on pp97/VCP. The
ryrosines
(Y796 and Y805) at the C-terminus of VCP are major phosphorylation sites that
may be
responsible for VCP roles in cell cycle regulation via heretofore
uncharacterized
molecular pathways (Eaerton et al.. 199 J. Biol. Chem. 169:11>>; Madeo et al.,
1998
~Llol. Biol. Cell 9:131 ).
Human 293 cells were co-transfected with (l) one of the HA-tagged
PTPH1 constructs (wildtype, D81 l~ or Y676FID811~) as described in E:camples 8-
10,
and (ii) either a wildtype VCP construct (VCPmyc) or a double mutant
(Y796F/Y805F)
VCP construct (VCPmyc-FF, L. Samelson, National Institutes of Health.
Bethesda.
Maryland) in which the two C-terminal tyrosine phosphorylation sites are
replaced with
phenylalanines. The VCP wildtype and mutant constructs were tagged with the
Vlyc
epitope recognized by monoclonal antibody 9E10 (American Type Culture
Collection.
Roclcville. Maryland). Co-transfected cells were lysed as described in
E.Yample 3 and
itamunoprecipitated with antibody 12C.~ (anti-HA) as described (Zhang et al.,
1997 J.
Biol. Chem: ?: ?:?7281). .
Electrophoretically resolved and blotted components were then probed
with anti-mvc antibody 9E10 to identify VCP proteins that co-precipitated with
(I.e..
were "upped" by) the PTPH1 protein. or with anti-H~ to confirm the presence of
~ PTPH1 proteins in immunoprecipitated material. Wildtype and mutant PTPH1
proteins
were e:cpressed at comparable levels. as were the two forms of VCP. The
PTPH11Y6 7 6F,'D811 _~) double mutant trapped wildrype VCP efficiently. but
did not
trap the double mutant VCP. which lacks two C-terminal tyrosine
phosphorylation sites
CA 02375145 2001-11-23
WO 00/75339 PCT/US00/14211
77
(Figure 9). tllso. neither wildtype PTPH1, nor the single mutant
PTPH1(D811?.),
effectively trapped VCP (Figure 9).
E~.:WLPLE 1?
SELECTIVE DEPHOSPHOItYL.~TION OF VCP BY PTPH1
In this e:cample, the effect of PTPH1 on the phosphorylation state of
VCP was e:camined. Stable ~T~ cells, transfected with and expressing full
length
wildtype PTPH1 under control of a tetracycline-repressible promoter in the
presence or
absence of tetracycline. were pretreated with 1 rWI vanadate for 1 hour prior
to lysis.
and VCP was immunoprecipitated using rabbit CS>; l antiserum. Lysates and
immunoprecipitates were prepared according to st~~d Precedttres as described
above
in Example 3. except cells were Iysed in RIPA buffer (i~1P40 buffer
supplemented with
1% sodium deo:rycholate and 0.1% SDS) instead of ~1P40 buffer. Under
conditions
permissive for PTPH1 e:cpression ('), a three- to five-fold decrease in the
1~ phosphotvrosine level of VCP was observed. relative to that seen when PTPHl
expression was repressed (-) (Figure 10A).
Lysates from the ~T~ t~fec'ants were also immunoprecipitated
with anti-phosphotyrosine antibody PT66 (Sigma, St_ Louis. MO) to obtain a
representative sample of tyrosine-phosphorylated proteins from cells cultured
in the
presence (~-) or absence (-) of PTPH1 e.~cpression (Figure 10B). Immunoblot
analysis of
these immunoprecipitates with antibodies specific for VCP revealed
dramatically
reduced levels of VCP among tyrosine-phosphoryiated proteins
immunoprecipitated
from cells in which PTPH1 expression was induced (~-) relative to uninduced
controls (-
(Figure 10B). The apparently selective dephosphorylation of VCP by PTPH1 in
these
?5 cells was also shown by assessing the effect of PTPH1 induction on the
decree of
tyrosine phosphorylation of a distinct tyrosine-phosphoryiated protein. the
kinase F ~K.
Induction of PTPH1 expression did not cause a corresponding decrease in the
level of
F ~K that was immunoprecipitated by anti-phosphotyrosine antibodies (Figure I
OB).
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78
The effects of induced PTPH1 expression on total tyrosme-
phosphorylated protein pools were also compared in PTPH1-transfected VIH~T3
cells
down under normal conditions ("untreated', under serum starvation by
cultivation in
DhfEVI containing 0.~% FBS for 16 hours (''starved'' or following insulin
stimulation
of starved cells by 10 u~/ml insulin (Roche Molecular Biochemicals.
Indianapolis. N)
for 10 minutes. Aliquots of total cell lysates were electrophoretically
resolved, blot
transferred to Immobilon-PT''' and probed with a mixture of two HRP-conjugated
anti-
phosphotyrosine antibodies. PY'_'0 (Transduction Laboratories, Le:cington, KY)
and
4610 (Upstate Biotechnology Inc., Lake Placid, ~ diluted according to the
suppliezs'
recommendations. followed by ECL detection (Amersham, Cleveland. OH). The
induction of PTPH1 overexpression failed to alter the global pattern of
protein tyrosine
phosphorylation in randomly bowing ("unu'eated'~, starved or insulin-
stimulated cells
(Figure 11).
1~ Those skilled in the art will recognize. or be able to ascertain, using no
more than routine experimentation, many equivalents to the specific
embodiments of
the invention described herein. Such equivalents are intended to be
encompassed by the
following claims. Also. it will be appreciated that. although specific
embodiments of
the invention have been described herein for the purpose of illustration,
various
modifications may be made without deviating from the spirit and scope of the
invention. Accordingly. the present invention is not limited except as by the
appended
claims.
