From the Cell Regulation Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore
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ABSTRACT |
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We have examined the in vivo activity
of receptor-like protein-tyrosine phosphatase (PTP
) toward
p59fyn, a widely expressed Src family kinase. In a coexpression
system, PTP
effected a dose-dependent tyrosine
dephosphorylation and activation of p59fyn, where maximal
dephosphorylation correlated with a 5-fold increase in kinase activity.
PTP
expression resulted in increased accessibility of the
p59fyn SH2 domain, consistent with a PTP
-mediated
dephosphorylation of the regulatory C-terminal tyrosine residue of
p59fyn. No p59fyn dephosphorylation was observed with
an enzymatically inactive mutant form of PTP
or with another
receptor-like PTP, CD45. Many enzyme-linked receptors are complexed
with their substrates, and we examined whether PTP
and
p59fyn underwent association. Reciprocal immunoprecipitations
and assays detected p59fyn and an appropriate kinase activity
in PTP
immunoprecipitates and PTP
and PTP activity in
p59fyn immunoprecipitates. No association between CD45 and
p59fyn was detected in similar experiments. The PTP
-mediated
activation of p59fyn is not prerequisite for association since
wild-type and inactive mutant PTP
bound equally well to
p59fyn. Endogenous PTP
and p59fyn were also found in
association in mouse brain. Together, these results demonstrate a
physical and functional interaction of PTP
and p59fyn that
may be of importance in PTP
-initiated signaling events.
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INTRODUCTION |
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Members of the Src family of tyrosine kinases have been implicated
in a variety of physiological and pathophysiological processes. These
include mediating mitogenic responses initiated by growth factor
receptors, control of cellular architecture through cytoskeletal reorganization, the UV and stress response, mitotic functions, and the
induction of tumors (for review, see Ref. 1). While the biological
roles of the Src family kinases are not known, it is well established
that the activities of these kinases are regulated, in part, by the
phosphorylation state of the negative regulatory tyrosine residue
corresponding to Tyr-527 of p60c-src (reviewed in
Refs. 2 and 3). Phosphorylation of this residue by Csk or Csk-like
kinases represses catalytic activity (4). Preventing phosphorylation of
this residue either by association with the polyoma virus middle
T-antigen or by mutation to phenylalanine or dephosphorylation of this
residue by protein-tyrosine phosphatases results in increased catalytic
and transforming activity (5-10). The identity of such phosphatases is
by and large unknown. As mentioned above, the hematopoietic cell
protein-tyrosine phosphatase (PTP)1 CD45 regulates the
phosphorylation state and activity of p56lck and p59fyn
in T cells (11-15). Presumably, there are other PTPs that regulate the
Src family kinases in cells lacking CD45. One possible candidate is
PTP, a receptor-type PTP.
PTP is a widely expressed protein that differs from most other
receptor-like PTPs in having a very short extracellular domain with no
adhesion motifs (16-19). Overexpression of PTP
leads to cell
transformation and to neuronal differentiation in rat embryo fibroblasts and in P19 carcinoma cells, respectively (20, 21). This is
similar to the actions of overexpressed epidermal growth factor
receptor in A431 and PC12 cells (22, 23), suggesting that PTP
may
normally play a role in stimulating cell proliferation. The
intracellular mediators of PTP
signaling are not known. The tyrosine
kinase pp60c-src is a candidate PTP
substrate
since PTP
overexpression in rat embryo fibroblasts and P19 cells
results in pp60c-src dephosphorylation and
activation (20, 21). PTP
may also exert some of its cellular effects
through its ability to bind the adaptor protein Grb2 (24-27).
Downstream components in a PTP
signaling pathway may include
mitogen-activated protein kinase and the transcription factor c-Jun,
both of which are activated in PTP
-overexpressing rat embryo
fibroblast cells (28). Whether PTP
mediates the dephosphorylation of
other cellular proteins besides pp60c-src is
unknown.
The similar structure and mode of regulation of Src family kinases
suggest that other members of this family may be PTP substrates. The
identification of PTP
substrates is an important step in elucidating
the biological role of PTP
. In this study, we have investigated the
action of PTP
toward p59fyn, prompted by a combination of
reasons. First, besides pp60c-src, only the Src
family kinases p59fyn and p62yes share a broad
expression pattern with PTP
(1). In addition, PTP
is highly
expressed in brain (18, 29), and PTP
, pp60c-src,
and p59fyn are implicated in or associated with certain
neuronal cell functions including neuronal differentiation (PTP
(21,
30) and pp60c-src (31, 32)), axonal growth
(pp60c-src (33) and p59fyn (34)),
myelination (p59fyn (35)), and spatial learning and memory
(p59fyn (36)). Second, together with
pp60c-src, p59fyn is well defined in terms
of its cellular actions. While studies in mutant mice show that Src and
Fyn kinases have a high degree of functional redundancy (37),
nevertheless, they also have specific and distinct functions (for
example, in cytoskeletal organization (38) and adhesion
molecule-directed axonal growth (33, 34)). It is thus important to
define which specific intermediate signaling molecules may mediate a
spectrum of PTP
-directed cellular events.
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EXPERIMENTAL PROCEDURES |
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Expression Plasmids--
Numbering of the PTP amino acid
sequence is according to Krueger et al. (17). The expression
vector pXJ41-PTP
-neo, encoding full-length PTP
, has been
described (20). The mutagenesis altering the essential cysteine
residues to serine residues in the active sites of PTP
(C414S/C704S)
has been described (39), and a fragment of this cDNA was used to
replace the corresponding piece of wild-type PTP
cDNA to produce
pXJ41-PTP
(C414S/C704S)-neo. Vectors expressing VSVG-tagged versions
of PTP
were constructed as follows. Primers (with PacI
sites added) corresponding to the amino acid sequences RVGIHL and
MNRLGK found at either end of a 29-amino acid C-terminal fragment of
VSVG (40) were used in a polymerase chain reaction with VSVG cDNA
(a gift from Dr. S. H. Wong) as template. The primer sequences were 5'-GCGGTTAATTAACCGAGTTGGTATTTATCTT-3' (forward) and
5'-GCGGTTAATTAACTTTCCAAGTCGGTTCAT-3' (reverse). The amplified fragment was inserted into a unique PacI site in the PTP
cDNAs of pXJ41-neo, permitting the expression of PTP
proteins
with a VSVG tag at amino acid 16 in the extracellular region. Plasmid containing CD45 cDNA (pAW-HCLA) was a gift of Dr. G. Koretzky. The
CD45 cDNA insert was removed with HindIII and subcloned
into the HindIII site of pXJ41-Hy (pXJ41 containing the gene
conferring hygromycin resistance). fyn cDNA was isolated
from a human fetal brain cDNA library in
gt11
(CLONTECH, Palo Alto, CA) and subcloned into the
EcoRI site of pXJ41-neo. Murine neuronal c-src
cDNA (NcoI fragment in pGEM5Z(+); Promega) was a
gift of Dr. P. Bello. The c-src cDNA insert was removed
with SphI and SacI and blunt end-ligated into the
EcoRI site of pXJ41-neo.
Cell Culture and Transient Transfections-- COS-1 cells were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin in an atmosphere of 5% CO2 at 37 °C. COS-1 cells at 50-70% confluency (100-mm dishes) were transfected with plasmid DNA by liposome-mediated transfection with 30 µl (1 mg/ml) of LipofectinTM or LipofectAMINETM reagent (Life Technologies, Inc.) for 5-6 h as described by the manufacturer and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for an additional 18-40 h prior to harvesting. The empty expression plasmid pXJ41-neo was used to normalize the amount of DNA in each transfection.
Western Blots and Immunoprecipitations from Transfected
Cells--
In experiments that did not involve the association of
PTP (or CD45) and p59fyn, cell extracts were prepared by
lysing cells either in buffer A (50 mM Tris-Cl (pH 7.2),
150 mM NaCl, 0.2 mM
Na3VO4, 1% Triton X-100, 10 µg/ml aprotinin,
and 0.1 mM phenylmethylsulfonyl fluoride) (see Figs. 2 and
4C) or in modified radioimmune precipitation assay buffer
(10 mM sodium phosphate (pH 7.0), 150 mM NaCl,
1 mM EDTA, 50 mM NaF, 0.1 mM
Na3VO4, 1% Nonidet P-40, 0.1% SDS, 1% sodium
deoxycholate, 10 µg/ml aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride) (see Figs. 1 and 3) for 60 min at
4 °C. Cytosol and Triton X-100-solubilized membrane fractions of
cells were obtained as described (20), essentially involving initial
lysis of cells by sonication in buffer A without Triton X-100. In
experiments involving co-immunoprecipitation of PTP
(or CD45) and
p59fyn, cell lysates were prepared in 10 mM Tris-Cl
(pH 7.2), 150 mM NaCl, 1 mM EDTA, 1% Brij 96, 10 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride (see Figs. 5-7). Lysates were clarified by centrifugation, and protein content was determined by Bradford analysis (41). Protein
extracts were separated by SDS-polyacrylamide gel electrophoresis on a
7 or 9% gel and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were immunoblotted with anti-PTP
antiserum 3897 (raised against a glutathione
S-transferase/PTP
-D2 fusion protein (39) containing the
second catalytic domain and C-terminal tail region of PTP
) followed
by goat anti-rabbit IgG conjugated to peroxidase (Sigma) or with
anti-VSVG (Sigma), anti-p59fyn (Transduction Laboratories),
anti-pp60c-src (Oncogene Science Inc.), or
anti-CD45 9.4 (Dr. G. Koretzky) monoclonal antibodies followed by goat
anti-mouse IgG conjugated to peroxidase (Sigma) or
peroxidase-conjugated anti-phosphotyrosine antibody (Transduction
Laboratories). Immunoblots were developed using the ECL system
(Amersham Pharmacia Biotech). Immunoprecipitations of cell lysates were
carried out using 250-600 µg of protein. For immunoprecipitation of
p59fyn, either anti-Fyn antiserum (a gift of C. Rudd; see Fig.
1) or anti-Fyn polyclonal antibody (FYN3-G, Santa Cruz Biotechnology, Inc.) was added to the cell lysates and incubated for 60-120 min at
4 °C. For pp60c-src and VSVG-PTP
immunoprecipitations, anti-pp60c-src or anti-VSVG
monoclonal antibodies were added to the cell lysates and incubated for
60 min at 4 °C, followed by incubation with rabbit anti-mouse IgG
(Dako Corp.) for another 60 min at 4 °C. Protein A cell suspension
(Sigma) was then added and mixed at 4 °C for 1 h. The
immunoprecipitates were washed three times each in the respective cell
lysis buffer (see above) and once in either 2× kinase assay buffer or
phosphatase assay buffer (see below). Immunoblot analysis of the
immunoprecipitated proteins was as described above.
Immunoprecipitations from Mouse Brain Lysate--
Whole brain
from an adult BALB/c mouse was homogenized in a hand-held Wheaton
homogenizer in 6-8 ml of 10 mM Tris-Cl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1% Brij 96, 10 µg/ml
aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. After
incubation at 4 °C for 90 min, the lysate was clarified by
centrifugation, and the protein content was determined. For
immunoprecipitations, 1 mg of lysate was diluted a further 4-fold in
homogenization buffer and then incubated with anti-PTP (3680, raised
against a peptide comprising the C-terminal 18 amino acids of PTP
),
anti-p59fyn (FYN3, Santa Cruz Biotechnology, Inc.), or anti-Csk
(C-20, Santa Cruz Biotechnology, Inc.) polyclonal antibodies. In some
experiments, anti-PTP
antibody 3680 was blocked before
immunoprecipitation by preincubation with recombinant purified
PTP
-D2 polypeptide (amino acids 485-774) for 45 min at 4 °C. The
brain lysate was incubated with the above antibodies for 16 h at
4 °C, and then Protein G PLUS/Protein A-agarose (Calbiochem) was
added, and incubation was continued for 60 min at 4 °C. The
immunoprecipitates were washed three times each in lysis buffer
containing Brij 96 and once in lysis buffer without detergent, resolved
by 8.5% SDS-polyacrylamide gel electrophoresis, and transferred to
polyvinylidene difluoride membranes. Membranes were immunoblotted with
anti-PTP
antibody 3897 or 3680 followed by goat anti-rabbit IgG
conjugated to peroxidase. Immunoblots were developed using the
SuperSignal chemiluminescence system (Pierce).
Accessibility of the p59fyn SH2
Domain--
Synthetic peptides with the sequence TSTEPQYQPGENL,
representing the sequence surrounding Tyr-527 of
pp60c-src, were made using either phosphotyrosine
or tyrosine at the appropriate step and were purified by high pressure
liquid chromatography (Biotechnology Center, National University
of Singapore). The phosphopeptide or peptide was covalently
coupled to CNBr-activated Sepharose-4B (Amersham Pharmacia Inc.) and
added to 300 µg of whole cell lysates (prepared with radioimmune
precipitation assay buffer as described above) of COS-1 cells
transfected with p59fyn cDNA alone or in combination with
PTP cDNA. After incubation for 2 h at 4 °C, the
Sepharose beads were washed twice each with radioimmune precipitation
assay buffer in the presence and absence of SDS/sodium deoxycholate,
respectively, and resolved by electrophoresis on a 10%
SDS-polyacrylamide gel. Immunoblot analysis was as described above.
Kinase Assays--
The kinase assays were performed in 20-µl
reactions containing 10 mM Pipes (pH 7.0), 5 mM
MnCl2, 0.5 mM dithiothreitol, 0.25 mM Na3VO4, and 5 µCi of
[-32P]ATP at 37 °C for 10 min. The reactions were
stopped with sample loading buffer, heated at 100 °C for 5 min,
resolved by 9% SDS-polyacrylamide gel electrophoresis, transferred to
polyvinylidene difluoride membranes, and autoradiographed.
Phosphatase Assays-- In experiments where PTP activity was measured, Na3VO4 was omitted from the cell lysis buffer. PTP activity was measured at 30 °C in reactions containing 50 mM Mes (pH 6.0), 0.5 mg/ml bovine serum albumin, 0.5 mM dithiothreitol, and 2.5-5 µM phosphotyrosyl-RR-Src peptide (RRLIEDAEY(P)AARG, corresponding to the sequence encompassing Tyr-416 of pp60c-src and phosphorylated as described (39)). The specific activity of the substrate ranged between 3000 and 4000 cpm/pmol of RR-Src. The reaction was carried out for 3 min unless otherwise indicated.
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RESULTS |
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PTP Effects Dephosphorylation of pp60c-src and
p59fyn--
The ability of PTP
to dephosphorylate
pp60c-src and p59fyn was assessed in COS-1
cells cotransfected with both kinases. Anti-phosphotyrosine probing of
p59fyn and pp60c-src immunoprecipitates
demonstrated that PTP
expression resulted in tyrosine
dephosphorylation of both kinases (Fig.
1, A and B). Blotting of these cell lysates with anti-cst.1, an antibody that recognizes both p59fyn and pp60c-src
equally well (42), showed that similar amounts of these kinases were
expressed with PTP
(data not shown). As p59fyn represents a
novel potential substrate for PTP
, we characterized the catalytic
action of PTP
toward p59fyn in more detail. The extent of
p59fyn dephosphorylation increased as increasing amounts of
PTP
cDNA were transfected, reaching a plateau of 80-90%
tyrosine dephosphorylation (Fig.
2A). Dephosphorylation was
completely dependent on the catalytic activity of PTP
since a PTP
mutant, PTP
(C414S/C704S), in which the essential cysteine residues
in the active sites of both catalytic domains of PTP
were mutated to
serine residues, was unable to effect p59fyn dephosphorylation
(Fig. 2A).
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Coexpression of PTP and p59fyn Results in
p59fyn Activation--
Besides tyrosine dephosphorylation
of p59fyn, the coexpression of PTP
resulted in kinase
activation of p59fyn. As shown in Fig. 2B, when
p59fyn immunoprecipitates from cells coexpressing PTP
were
used in an immunocomplex kinase assay, increasing p59fyn
autophosphorylation was observed with increasing expression of PTP
.
A maximum 5-fold increase in kinase activity was obtained (Fig.
2B, lanes 5 and 6) at the same
PTP
/p59fyn cDNA ratio observed to give maximal
p59fyn dephosphorylation (Fig. 2A). No increase in
the kinase activity of p59fyn was measured when catalytically
inactive PTP
was expressed with p59fyn (data not shown).
p59fyn Dephosphorylation and Activation Are Accompanied
by Increased SH2 Domain Accessibility--
Dephosphorylation of the
C-terminal tyrosine residue of Src family kinases is linked to kinase
activation (5-9) and correlates with increased c-Src SH2 domain and
Lck SH2 domain accessibility during mitosis and T cell activation,
respectively (43, 44). These observations support a model of kinase
activation where disruption of an intramolecular association between
the C-terminal phosphotyrosyl peptide and the SH2 domain of the kinase
results in catalytic activation as well as novel interactions of the
SH2 domain with other phosphotyrosyl proteins (2, 3). The effect of
PTP on p59fyn SH2 domain accessibility was examined by
determining the ability of p59fyn to bind to a synthetic
phosphopeptide representing the C-terminal Tyr-527 peptide of
pp60c-src. This peptide is identical to the
C-terminal sequence surrounding Tyr-531 of p59fyn except for
the replacement of alanine in position 2 with serine (45). As shown in
Fig. 3, PTP
-induced dephosphorylation
of p59fyn (Fig. 3C) correlated with a 3-fold
increase in the amount of Fyn protein precipitated from cell lysates
with the Src Tyr-527 phosphopeptide coupled to Sepharose beads (Fig.
3D, bottom panel). A control precipitation using
an unphosphorylated Src Tyr-527 peptide-Sepharose conjugate contained
barely detectable but equivalent amounts of Fyn protein from
p59fyn- and PTP
/p59fyn-expressing cells (Fig.
3D, top panel). While the site(s) of
p59fyn dephosphorylation remain to be mapped, the increased
p59fyn catalytic activity and SH2 availability for binding are
consistent with a PTP
-mediated dephosphorylation of the C-terminal
Tyr-531 of p59fyn.
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Specificity of PTP in Effecting p59fyn
Dephosphorylation and Activation--
To examine whether the
PTP
-mediated p59fyn dephosphorylation was a specific effect
of PTP
or merely reflected a nonspecific increase in PTP activity at
the cell membrane, the tyrosine phosphorylation state of p59fyn
was analyzed upon coexpression with another receptor-like PTP, CD45.
CD45 is a hematopoietic cell-specific molecule required for T and B
cell activation (46-48) and can dephosphorylate the Src family kinases
p59fyn and p56lck in T cells (11-15) and
p62lyn in B cells (49). Fractionation of transfected COS cells
into solubilized membrane and cytosol followed by Western blotting showed that, like PTP
(Fig.
4A, middle panel),
CD45 is localized to membranes (bottom panel). As expected,
a majority of the Fyn protein was also associated with membranes (Fig.
4A, top panel). Elevated membrane PTP activity
was measured in PTP
- or CD45-expressing cells (Fig. 4B),
demonstrating that both receptor PTPs are enzymatically active and that
comparable levels of phosphatase activity are seen in both cell types.
However, immunoprecipitated p59fyn was
tyrosine-dephosphorylated in PTP
-expressing cells (Fig. 4C, compare lanes 1 and 2), whereas no
reduction in the tyrosine phosphate content of p59fyn from
CD45-expressing cells was detected (compare lanes 1 and 3). Even when the amount of CD45 cDNA was increased
4-fold over that used for the experiments shown in Fig. 4, no
dephosphorylation of coexpressed p59fyn was observed (data not
shown). This is similar to another report that CD45 cannot effect
p56lck dephosphorylation when expressed in non-lymphoid cells
(15). The p59fyn dephosphorylation observed in the presence of
PTP
, but not CD45, correlated with elevated kinase activity of Fyn
in PTP
-expressing cells (as described above) and no alteration in
the kinase activity of p59fyn from CD45-expressing cells (data
not shown). Thus, increased membrane PTP activity is not in itself
sufficient to effect p59fyn dephosphorylation and activation,
indicating that the PTP
-mediated dephosphorylation and activation of
p59fyn reflect a specific effect of PTP
.
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Association of PTP and p59fyn--
To further
investigate the interaction of PTP
and p59fyn, we examined
whether these two proteins underwent any form of association. To enable
immunoprecipitation of PTP
, a 29-amino acid epitope from VSVG was
inserted into the PTP
extracellular region to create VSVG-PTP
.
Cells expressing p59fyn alone or in conjunction with
VSVG-PTP
were lysed under mild detergent conditions, and PTP
was
immunoprecipitated with anti-VSVG antibodies. Probing of the
immunoprecipitates with anti-VSVG antibodies detected two forms of
VSVG-PTP
: a broad diffuse band migrating at ~130 kDa, consistent
with the size of wild-type glycosylated PTP
, and a sharper band at
~100 kDa, consistent with the size of incompletely glycosylated
PTP
(50) (Fig. 5A,
top panel, lanes 7 and 8). Probing of
these samples and whole cell lysates with anti-p59fyn
antibodies revealed about equal amounts of Fyn protein in cell lysates
expressing p59fyn alone or together with VSVG-PTP
(Fig.
5A, bottom panel, lanes 2 and
4) and a significant amount of p59fyn in the
anti-VSVG immunoprecipitate from the PTP
/p59fyn-coexpressing
cells (lane 8). The presence of this p59fyn in the
VSVG immunoprecipitate was due to its interaction with VSVG-PTP
since only a weak p59fyn signal (likely due to nonspecific
sticking) was detected in anti-VSVG immunoprecipitates prepared from
cells expressing p59fyn in the absence of VSVG-PTP
(Fig.
5A, bottom panel, lane 6). In
addition, in vitro assay of kinase activity in the anti-VSVG immunoprecipitates detected enhanced phosphorylation of a protein (Fig.
5B, lane 5) that comigrated with
autophosphorylated p59fyn produced by in vitro
kinase assay of an anti-Fyn immunoprecipitate from
p59fyn-expressing cells (lane 1). These and
subsequent experiments to examine the association of PTP
and
p59fyn were carried out with cell lysates prepared by
solubilization with Brij 96, a mild non-ionic detergent. The
association of PTP
and p59fyn was also detected in lysates
prepared in buffer containing Triton X-100 or in radioimmune
precipitation assay buffer, indicating that the interaction is also
stable in other non-ionic detergents and in ionic detergents (data not
shown). The above results indicate that p59fyn associates with
and can be co-immunoprecipitated with PTP
.
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Association of p59fyn and a Catalytic Mutant of
PTP--
The mechanism and temporal occurrence of PTP
and
p59fyn association (pre- or post-dephosphorylation) were
examined using a catalytically inactive form of PTP
. Mutation of the
essential cysteine residue in the conserved catalytic domain of PTPs
creates an enzymatically inactive PTP (51, 52), which has been shown to
bind to and "trap" phosphotyrosyl substrates (53-55). A
VSVG-PTP
double mutant in which the essential cysteine residues in
both the membrane proximal and distal catalytic domains were mutated to
serine residues (C414S/C704S) was produced. If association involves the
recognition of phosphotyrosine sites in p59fyn as a
pre-dephosphorylation event, then such interaction might be stabilized
and enhanced with enzymatically inactive PTP
. However, if
p59fyn dephosphorylation is prerequisite for
PTP
-p59fyn association, then the phosphatase-kinase complex
will not be formed with the enzymatically inactive mutant. No
dephosphorylation of p59fyn was detected upon coexpression with
mutant VSVG-PTP
(C414S/C704S) (data not shown, but see Fig.
2A). Nevertheless, p59fyn was detected in
immunoprecipitates of VSVG-PTP
(C414S/C704S) at a level equivalent to
that of p59fyn in immunoprecipitates of catalytically active
VSVG-PTP
(Fig. 7). Thus, a
conventional substrate-trapping technique fails to enhance
PTP
-p59fyn interaction, suggesting that additional or
alternative regions of these proteins are responsible for association.
Also, the finding that p59fyn and PTP
can associate
independently of PTP
activity, and thus in the absence of or prior
to dephosphorylation, indicates that PTP
is suitably positioned to
directly utilize p59fyn as a substrate.
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Association of Endogenous PTP with p59fyn--
The
above studies were carried out with COS cells ectopically expressing
PTP
and p59fyn. To determine if association occurred between
these proteins when present at endogenous levels, we examined whether
they could be co-immunoprecipitated from mouse brain lysates. Brain was
chosen as the tissue source because PTP
is highly expressed in brain (18, 29) and because neuronal functions of both PTP
(21, 30) and
p59fyn (34-36) have been reported. None of the PTP
antibodies available to us immunoprecipitated PTP
very efficiently,
based on comparisons with the level of PTP
detected by Western
blotting of brain lysates (data not shown). Some PTP
could be
precipitated (Fig. 8, lane 2)
with a polyclonal antibody (3680) raised against a synthetic peptide
corresponding to the C-terminal 18 amino acids of PTP
, and specific
immunoprecipitation of PTP
was blocked by preincubation of the
antibody with recombinant purified protein (PTP
-D2) comprising the
membrane distal catalytic domain and C terminus of PTP
(Fig. 8,
lane 1). PTP
was detected in p59fyn
immunoprecipitates (Fig. 8, lane 3), but not in
immunoprecipitates of Csk (lane 4), a non-receptor tyrosine
kinase structurally similar to p59fyn. The above
immunoprecipitations were probed with anti-PTP
antibody (3897)
raised against recombinant PTP
-D2. Stripping and reprobing of
lanes 1-4 with the anti-PTP
C-terminal antibody (3680, used for PTP
immunoprecipitations; see above) gave the same results (data not shown). This association of PTP
with p59fyn was
observed in repeated experiments and demonstrates that these proteins are physiologically associated.
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DISCUSSION |
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We have confirmed that PTP dephosphorylates
pp60c-src in vivo and found that PTP
can also dephosphorylate the related Src family kinase p59fyn.
The concomitant increase in p59fyn kinase activity and increase
in accessibility of the p59fyn SH2 domain are consistent with a
PTP
-mediated dephosphorylation of the C-terminal tyrosine residue
thought to be critical in regulating the activity of p59fyn. In
contrast, the expression of the receptor-like PTP CD45 in this system
did not result in p59fyn dephosphorylation or activation,
demonstrating a specific action of PTP
on p59fyn.
Previously, PTP
activity has mainly been assayed for artificial substrates in vitro using immunoprecipitated PTP
or
bacterially expressed forms of cytosolic PTP
(24, 39, 56, 57). This characterization of a cellular assay of PTP
activity will be useful
in the future assessment of the effects of various structural mutants
of PTP
on its enzymatic function.
An important question regarding the activation of Src family kinases by
PTP is whether these kinases are directly dephosphorylated by PTP
and thus represent PTP
substrates. Potential PTP
substrates might
be complexed with the phosphatase, as is the case with many tyrosine
kinases and their substrates, or with CD45 and its substrates in T
(58-62) and B (63) cells. Co-immunoprecipitation experiments with
transfected cells demonstrated that PTP
and p59fyn were
consistently found to be associated with one another. This was not
merely an artifact of heterologous expression of these proteins since
CD45 and p59fyn were not found in association under the same
experimental conditions. Likewise, what appears to be an incompletely
glycosylated (and thus perhaps inappropriately localized) form of
PTP
did not associate with p59fyn. In addition, PTP
was
detected in p59fyn immunoprecipitates from mouse brain.
Although PTP
-p59fyn association is suggestive of a direct
enzyme-substrate relationship, at present it is unclear whether these
physical and functional actions of PTP
are linked. If so, two
possibilities are that association enables the subsequent
dephosphorylation and activation of p59fyn or that activated
p59fyn directly or indirectly modifies PTP
to promote
PTP
-p59fyn association, perhaps after tyrosine
phosphorylation of the phosphatase creates p59fyn-binding
sites. Our evidence supports the former scenario since the association
of a catalytically inactive form of PTP
with p59fyn
indicates that this physical interaction does not require prior dephosphorylation/activation of p59fyn. We are generating
truncated forms of PTP
to define the region(s) involved in
p59fyn binding. Regions of p59fyn that associate with a
variety of other signaling molecules include the SH3 and SH2 domains
(for examples, see Refs. 64-67) and the unique N-terminal region (68,
69). A proline-rich sequence similar to the consensus sequence for SH3
binding (70-72) is found in PTP
(RKYPPLP, amino acids 188-194). As
PTP
is tyrosine-phosphorylated in the cell (25, 26), this could
provide sites for SH2 binding. Alternatively, PTP
-p59fyn
association may occur through other regions or intermediary
proteins.
Besides the association with p59fyn described here, PTP can
associate with the adaptor molecule Grb2 (24-27). The latter complex is formed upon phosphorylation of a tyrosine residue in the tail region
of PTP
and binding by the SH2 domain of Grb2. A direct or indirect
association also occurs between the C-terminal SH3 domain of Grb2 and a
non-proline-rich region near the active site of the membrane proximal
catalytic domain of PTP
, but is not observed in the absence of the
PTP
-Grb2(SH2) interaction. One effect of PTP
-Grb2(SH3) binding is
postulated to be the inhibition of the catalytic activity of PTP
through the obstruction of substrate binding (26). Tyrosine
phosphorylation of PTP
can be catalyzed by
pp60c-src (24), and it will be of interest to see
if p59fyn can phosphorylate PTP
at the same site to result
in Grb2 binding. We have observed enhanced tyrosine phosphorylation of
PTP
upon coexpression with
p59fyn,2 although the
site(s) of phosphorylation is unknown. If this phosphorylation occurs
at the appropriate C-terminal site, PTP
-catalyzed activation of
pp60c-src and/or p59fyn would activate
kinase-mediated downstream signaling events while also permitting the
feedback inhibition of PTP
by effecting Grb2 binding. Regardless of
whether p59fyn activation and Grb2 binding are linked, it will
be of interest to see whether p59fyn and Grb2 binding to PTP
can occur on the same PTP
molecule or are mutually exclusive.
Previous studies have suggested that the cell transformation and
retinoic acid-induced neuronal differentiation observed in certain cell
types upon PTP expression may be mediated through pp60c-src (20, 21). Here we have provided further
evidence that PTP
is a physiological regulator of the Src family
kinases and, in particular, that p59fyn is a direct in
vivo substrate of PTP
. As with pp60c-src,
prevention of C-terminal Tyr-531 phosphorylation of p59fyn (by
mutation of this tyrosine to phenylalanine) results in an oncoprotein,
which upon overexpression transforms rodent fibroblasts (73). The
altered phenotype of PTP
-expressing cells may be due to the
increased kinase activity of p59fyn. Alternatively,
PTP
-induced transformation and differentiation may be a consequence
of the combined or synergistic effect of increased p59fyn and
pp60c-src catalytic activity. The association of
PTP
with p59fyn in brain, in conjunction with the presence
of both proteins in neuronal cells such as cerebellar granule cells
(29, 74, 75) and dorsal root ganglia (34, 76), suggests that
p59fyn could be a component of as yet unknown PTP
signaling pathways of neuronal development.
Dephosphorylation and activation of Src family kinases have been
demonstrated or implicated in studies of the signaling pathways of two
receptor-like PTPs, PTP and CD45. It is conceivable that these and
other non-adhesion receptor-type PTPs share a common mode of signaling,
with specificity determined by the respective spatial and temporal
patterns of gene expression of the receptor-type PTPs and Src family
kinases.
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ACKNOWLEDGEMENTS |
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We thank G. Koretzky for the CD45 cDNA and 9.4 antibody, C. Rudd for anti-p59fyn antibody, S. Courtneidge for cst.1 antibody, P. Bello for the pp60c-src cDNA, S. H. Wong for the VSVG cDNA, and W.-P. Yu for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the National Science and Technology Board of Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 65-874-3742;
Fax: 65-779-1117; E-mail: mcbcp{at}imcb.nus.edu.sg.
1 The abbreviations used are: PTP, protein-tyrosine phosphatase; VSVG, vesicular stomatitus virus glycoprotein; Pipes, 1,4-piperazinediethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid.
2 V. Bhandari, K. L. Lim, and C. J. Pallen, unpublished observations.
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REFERENCES |
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