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INTRODUCTION |
Within the protein-tyrosine phosphatase
(PTP)1 superfamily, PTP-PEST
is a ubiquitously expressed cytoplasmic enzyme with the conserved
catalytic domain located at the amino terminus of the protein (1).
PTP-PEST also contains a long carboxyl-terminal tail carrying several
signal transduction motifs. Among these motifs, two classical
proline-rich stretches have been shown to interact with the SH3 domains
of Grb2, p130Cas, Hef1/Cas-L, Sin/Efs, and Csk (2-5). In
addition, a novel and non-classical polyproline region in PTP-PEST has
been implicated in the binding of paxillin and Hic-5 (6-9). Finally,
the PTB domain of the Shc proteins has been reported to interact with a
NPLH sequence of PTP-PEST in a phosphotyrosine-independent manner (10).
A common theme among PTP-PEST-associated proteins is their implication
in the actin cytoskeleton and in focal adhesion signaling, suggesting a
regulatory role for PTP-PEST in these specialize cellular structures
(11).
The identification of p130Cas as a substrate of PTP-PEST
using catalytically inactive substrate trapping mutants of the enzyme was a major step in the elucidation of some of the biological functions
of this protein (3, 5, 12). Although the PTP domain of PTP-PEST is
sufficient to recognize the p130Cas phosphorylated on
tyrosine residues in vitro, an additional level of
specificity is mediated via a SH3 domain-dependent
association in vivo (3, 5). In PTP-PEST 
/ cells,
p130Cas is hyperphosphorylated and the cells exhibit
impaired migration on a fibronectin matrix, presumably due to their
inability to turn over focal adhesions and actin stress fibers (13).
Additionally, in Rat-1 fibroblast cell lines overexpressing PTP-PEST,
p130Cas was demonstrated to be constitutively
hypophosphorylated. In these cells, motility defects were also
observed and explained by the failure of these cells to form the
p130Cas·CrkII complex, which is required for
integrin-dependent migration (14). Recently, it has been suggested that
c-Abl, Shc, Pyk2, and Fak may well be additional substrates of PTP-PEST
(15-17).
PTP-PEST belongs to a subfamily of PTPs that includes PEP, PTP-HSCF,
and BDP (3, 18). These PTPs are grouped together because, in addition
to the homology in their PTP domains, they also contain a similar amino
acid motif at their complete carboxyl-terminal termed the CTH domain.
Previously, PTP-HSCF, a protein restricted to hemapoietic stem cells,
was shown to interact with the proline, serine,
threonine-rich phosphatase
interacting protein (PSTPIP) via its CTH domain
(18). The discovery that WASP, the protein encoded by the gene mutated
in the Wiskott-Aldrich syndrome, is a ligand for the SH3 domain of
PSTPIP creates an intriguing link between PSTPIP and actin
reorganization (19).
PSTPIP appears to be the mammalian homologue of the fission yeast
Schizosaccharomyces pombe CDC15p, a protein involved in the
organization of the cytokinetic cleavage furrow (20). It was reported
that PSTPIP associates with cortical actin during most of the cell
cycle and translocates at the cleavage furrow during cytokinesis (18).
Although the function of PSTPIP in the cleavage furrow can be thought
to be similar to the function of CDC15p in yeast, the role of PSTPIP at
the cortical actin during most of the cell cycle is completely unknown.
Several mammalian proteins sharing a similar domain organization as
PSTPIP have recently been identified. CIP4, PACSIN1/SyndapinI,
PACSIN2/Syndapin2, PACSIN3, and PSTPIP2/MAYP all have an amino terminus
homologous to CDC15p (21-26). The amino terminus of these proteins can
further be subdivided into a FCH (Fer kinase and
CIP4 homology) and a CDC15-like coiled-coil
domain (25). Recently, the FCH domain of CIP4 was shown to interact
with microtubules (27). Interestingly, all these PSTPIP-like proteins,
with the exception of PSTPIP2/MAYP, have a carboxyl-terminal SH3 domain
involved in the binding of WASP and/or N-WASP. Together, these proteins
form a family of distantly related members that could share overlapping
functions in actin-dependent biological events.
WASP, N-WASP, and WAVEs (also know as SCARs) proteins interact with and
activate the Arp2/3 actin nucleation activity (28, 29). These proteins
are inactive at the resting state because of an intramolecular
interaction involving the Arp2/3-binding site (known as the VCA or WCA
domain) and GTPase-binding domain (30). Studies to date have
demonstrated that the binding of GTP-loaded CDC42 and
phosphatidylinositol 4,5-biphosphate (PIP2) to N-WASP and
WASP is sufficient to release the inhibitory intramolecular interactions and activate these proteins (31-33). Furthermore, SH3
domain-containing proteins, such as Grb2, Nck, and WISH, can interact
with a proline-rich region found on the WASP-like proteins and
cooperate in their activation (34-36). The activation of the WASP
proteins lead to actin nucleation via the recruitment and activation of
the Arp2/3 complex, and to the formation of filipodia (N-WASP) and
lamelipodia (WAVE) (33). These events are important in the generation
of cell polarity, directed cell migration, vesicule transport and
endocytosis (37-39).
In PTP-PEST / cells, we previously noted the hyperphosphorylation
of PSTPIP and a defect in cytokinesis (13). This guided us to
investigate if PSTPIP can interact directly with PTP-PEST. In this
article, we report that PSTPIP and PTP-PEST associates in
vivo. The coiled-coil domain of PSTPIP and the CTH domain of PTP-PEST mediate this interaction. We also demonstrate that PTPIP becomes tyrosine phosphorylated in mouse fibroblasts following EGF or
PDGF treatment. This phosphorylation of PSTPIP is independent of Src
kinases since the PP2 inhibitor did not affect it. We provide evidence
that PTP-PEST negatively regulates the association of PSTPIP with SH2
domain-containing proteins by the dephosphorylation of PSTPIP on
tyrosine 344. In contrast, the association of WASP with PSTPIP was
demonstrated to be constitutive in vivo and independent of
PSTPIP tyrosine phosphorylation. However, PSTPIP creates a physical
link between PTP-PEST and WASP resulting in a specific PTP-PEST-mediated dephosphorylation of WASP.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
NIH 3T3, HER14, HEK293-T,
COS-1, and RAW 264.7 cell lines were grown in Dulbecco's modified
Eagle's medium. 70Z/3 and BaF/3 pro B-cells (obtained from Dr. A. Veillette, IRCM, Montréal) were grown in RPMI 1640. All cell
culture media were supplemented with 10% fetal bovine serum
(BIOSOURCE) and a mixture of
penicillin/streptomycin (Invitrogen). COS-1, NIH 3T3, and HER14 cell
lines were serum starved for 48 h in media containing 0.1% serum.
These cells were treated with either 25 ng/ml PDGF (Roche Molecular
Biochemicals), 100 ng/ml EGF (Sigma), 1 µM insulin
(Invitrogen) or media alone for 15 min prior to lysis. The PP2
inhibitor (Calbiochem) was used at 10 µM in Dulbecco's
modified Eagle's medium and was added to the cells 30 min prior EGF
stimulation. HEK293-T and COS-1 cells were transfected with 5-10 µg
of total plasmid DNA using the calcium phosphate precipitation method
as previously described (40).
Antibodies--
The polyclonal antibody against PSTPIP (number
2221) and its HRP conjugate were previously reported (13). The
anti-PTP-PEST (number 1075) was described (40). Two additional
polyclonal antibodies were generated in this study against PTP-PEST:
number 2528 (immunogen: GST-344-437) and number 2530 (immunogen:
GST-471-613). The anti-Flag M2 monoclonal antibody was obtained from
Sigma. The anti-c-Src, FAK, c-Cbl, c-Abl, and WASP were from Santa Cruz Biotechnologies. The anti-Fyn was a gift from Dr. A. Veillette. The
anti-Lyn was purchased from Pharmingen.
Plasmids and Constructions--
PTP-PEST, WT, and C231S, in the
expression vector pcDNA3.1 Zeo(+) were previously described.
PTP-PEST with a deleted Pro1 or Pro2 (reported in Refs. 3 and 6) were
subcloned from pACTAG into the NotI and XbaI
sites of pcDNA3.1 Zeo. PTP-PEST with a deleted CTH domain (
CTH)
was constructed by digesting the pcDNA3.1 Zeo PTP-PEST WT with
PstI and religation of the vector following gel purification
(QIAEX II, Qiagen). To construct pcDNA3.1 Zeo PTP-PEST C231S
Pro3-5, the PTP-PEST C231S cDNA was digested with
BamHI and ApaI and ligated in the same sites of
pcDNA3.1 Zeo. To construct EFGP PTP-PEST, the cDNA of PTP-PEST
was amplified by PCR using engineered oligonucleotides containing
BamHI and SalI restriction sites, and the
digested PCR product was ligated into pEGFP-C2 linearized with
BglII and SalI. EGFP PTP-PEST CTH was
generated by subcloning the BamHI and PstI
fragment of the PTP-PEST cDNA into BglII and
PstI sites of pEGFP-C1. Full-length PSTPIP in pRK5 Flag C
was previously described (18). PSTPIP was subcloned in pcDNA3.1 Zeo
in the NotI/XhoI sites. Tryptophan 232, tyrosine
344, and tyrosine 367 were mutated by strand overlap extension PCR
using oligonucleotides with point mutations as described in Ref. 6.
These procedures resulted in the following mutants: W232A (coiled-coil
domain mutant), Y344F, and Y367F. All the mutations were verified by
dideoxy sequencing using Sequenase (Amersham Bioscience, Inc.). The
plasmid constructions for the GST fusion proteins of the SH2 domain of
Crk, c-Abl, Fyn, and c-Src were obtained from Dr. M. Park (McGill
University). The SH2 domain of Nck was a gift from Dr. L. Larose
(McGill). pcDNA3.1 LCK, pSR
v-Src, pcDNA3.1 avian c-Src, and
Fyn were kind gifts from Dr. A. Veillette. pME Lyn was a kind gift from
Dr. T. Yamamoto (The University of Tokyo, Japan), c-Abl cDNA was a
gift from Dr. O. N. Witte (University of California, Los Angeles,
CA), and the cDNA of WASP was a kind gift of Dr. N. Lamarche (McGill).
Immunoprecipitation--
Cells were lysed in 1 ml of lysis
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
and 1% Nonidet P-40 supplemented with 1 mM sodium
orthovanadate, 5 mM NaF and a Complete protease inhibitor mixture (Roche Molecular Biochemicals) for 10 min on ice. For in
vivo substrate trapping, the lysis buffer was modified in its sodium orthovanadate concentration (0.1 mM instead of 1 mM) to avoid competition of the inhibitor with the C231S
mutants of PTP-PEST. Following growth factor treatments, NIH 3T3 and
HER14 cells were lysed in 1 ml of RIPA (40) for 10 min and diluted 1:1
with the Nonidet P-40 lysis buffer described above. The lysates were
harvested and cleared by centrifugation at 16,000 × g
at 4 °C. Aliquots of cell extracts (500-1000 µg of proteins) were
immunoprecipitated with 1-4 µl of polyclonal antibodies and 20 µl
of Protein-A agarose in a final volume of 1 ml for 90 min at 4 °C.
The immunoprecipitates were washed three times in 1 ml of lysis buffer,
resuspended in 40 µl of SDS sample buffer, and finally boiled for 5 min at 95 °C. Typically, a fourth of the immunoprecipitations (10 µl) were resolved by 7.5% SDS-PAGE and the proteins were transferred
to PVDF membranes (Immobilon-P, Millipore).
In Vitro Translation--
pACTAG PSTPIP was linearized with
XbaI and used as a template for in vitro
transcription using T7 RNA polymerase. Two microliters of the
transcription reaction were used to perform in vitro
translation in the presence of 35S-labeled methionine and
rabbit reticulocyte lysate (Promega). The in vitro
translated PSTPIP was assayed for binding to GST alone and GST PTP-PEST
fusion proteins as described below.
In Vitro Binding Assay Using GST Fusion Proteins--
GST fusion
proteins were expressed in exponentially growing Escherichia
coli DH5 by induction for 2 h with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. Bacteria were
harvested by centrifugation (typically 1.5 ml per binding assay) and
the fusion proteins were isolated on gluthatione-Sepharose as
previously described (3). Each fusion protein was incubated with the
appropriate cell extract in immunoprecipitation buffer for 90 min at
4 °C. The beads were washed three times with 1 ml of lysis buffer,
resuspended in 40 µl of SDS sample buffer, and boiled for 5 min at
95 °C. The GST pull-downs were resolved by 7.5% SDS-PAGE and the
proteins were transferred to PVDF membranes.
Metabolic Labeling and Phosphopeptide Maps--
Experiments were
performed as described in Refs. 41 and 42. Briefly, transfected
HEK293-T cells were rinsed twice and incubated in phosphate-free
Dulbecco's modified Eagle's medium (0.5% dialyzed fetal bovine
serum) for 1 h. The cells were then incubated with 0.25 mCi/ml
[32P]orthophosphate in phosphate-free Dulbecco's
modified Eagle's medium (0.5% dialyzed fetal bovine serum) for 4 h. The cells were rinsed twice with ice-cold 50 mM HEPES,
pH 7.5, and lysed as described in the immunoprecipitation section.
Cellular debris were removed by centrifugation and the PSTPIP proteins
were immunoprecipitated with 3 µl of the polyclonal antibody number
2221. The immunoprecipitated proteins were subjected to SDS-PAGE and
transferred to PVDF. The membranes were air-dried and autoradiographed.
The regions of PVDF containing PSTPIP or its mutants were cut and
digested with 10 µg of TPCK-treated trypsin for 16 h, followed
by the addition of 5 µg of TPCK-treated trypsin for 3 h. The
samples were rinsed with water and lyophilized. This procedure was
repeated three times. The samples were resuspended in
electrophoresis buffer (2.5% (v/v) formic acid and 7.8% (v/v) glacial
acetic acid) and spoted on cellulose-coated thin layer chromatography
(TLC) plates. The electrophoresis was performed at pH 1.9 in a HTLE
7000 apparatus. The samples were separated in a second dimension by
thin layer chromatography in TLC buffer (39% (v/v) 1-butanol, 30%
(v/v) pyridine, 6.1% (v/v) glacial acetic acid). The TLC plates were
dried and autoradiographed to visualize the phosphopeptides.
Western Blotting--
PVDF membranes were blocked in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween
20) containing either 1% bovine serum albumin or 5% nonfat dry milk
for 60 min. The primary antibodies were incubated with the blot in
blocking buffer for 60 min at room temperature or overnight at 4 °C.
The polyclonal anti-PSTPIP (number 2221), anti-PSTPIP-HRP (number
2221), and anti PTP-PEST (numbers 2528 and 2530) antibodies were used
at 1:5000 dilutions in TBS-T with 5% milk. The monoclonal antibodies
against phosphotyrosine, 4G10 and 4G10-HRP, were used at 1:5000
dilutions in TBS-T with 1% bovine serum albumin. The commercial
antibodies were diluted according to the manufacturers recommendations.
The membranes were washed two times for 5 min and two times for 10 min
with TBS-T and incubated when needed with the appropriate secondary antibody conjugated to horseradish peroxidase for 45 min. The blots
were washed extensively by repeated incubation in TBS-T and the bound
antibodies were detected using chemiluminescence (Renaissance,
PerkinElmer Life Sciences).
Immunofluorescence--
COS-1 cells were transfected to
transiently co-express EGFP PTP-PEST (WT or CTH) and PSTPIP. Cells
were serum starved overnight, fixed with 4% (w/v) of paraformaldehyde,
permeabilized, and blocked as described in Ref. 13. The anti-PSTPIP
antibody (number 2221) was used at a 1:1000 dilution in PBS containing
2% bovine serum albumin. The anti-rabbit antibody conjugated to TRITC
(Jackson ImmunoResearch Laboratories Inc.) was used at a 1:200
dilution. Cells were visualized with a Nikon Eclipse TE300 microscope
using a ×40 objective (Nikon). Pictures were acquired employing a
digital camera (model Quantix, Photometrics) and the MetaMorph software (Universal Imaging Corp.).
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RESULTS |
PSTPIP and PTP-PEST Expression Overlaps in Mouse Tissues and Cell
Lines--
We previously reported a defect in cytokinesis in addition
to the hyperphosphorylation of PSTPIP in PTP-PEST / cells (13). This guided us to investigate if PSTPIP can interact directly with
PTP-PEST. A graphical representation of PSTPIP and its mutant used in
this study is presented in Fig.
1A. PSTPIP contains an amino-terminal FCH domain, a coiled-coil domain, a SH3 domain, and two
putative tyrosine phosphorylation sites. We generated a polyclonal
antibody against the SH3 domain of PSTPIP (Fig. 1A). This
domain of PSTPIP has been reported to become tyrosine phosphorylated (19). Since several experiments using this antibody will aim at
characterizing the tyrosine phosphorylation level of PSTPIP and its
association with other proteins, we studied the anti-PSTPIP antibody in
more detail. As seen in Fig. 1B, the antibody can precipitate PSTPIP from NIH 3T3 fibroblasts and 70Z/3 pro-B cells (bottom panel), either unphosphorylated or highly tyrosine
phosphorylated because of pervanadate treatment of the cells prior to
lysis (upper panel). In addition, the antibody could
co-precipitate several tyrosine phosphorylated proteins together with
PSTPIP from pervanadate-treated NIH 3T3 and 70Z/3 cell lysates
(upper panel, lanes 4 and 8). The preimmune serum did not precipitate any PSTPIP nor any other
tyrosine-phosphorylated protein.
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Fig. 1.
PSTPIP and PTP-PEST are co-expressed in
various mouse tissues and cell lines. A, schematic
representation of PSTPIP and its mutants used in this study.
B, characterization of the antibody against PSTPIP. NIH 3T3
and 70Z/3 were treated or not with pervanadate and PSTPIP was
immunoprecipitated from the cell lysate with either the preimmune or
the anti-PSTPIP serum to ascertain that tyrosine phosphorylation of
PSTPIP does not affect recognition by the antibody. PSTPIP was
precipitated to the same extent in both cell lines (bottom
panel) and regardless of its phosphorylation state (top
panel). No signal was detected in the preimmune precipitations
demonstrating the specificity of the antibody. C, Western
blotting was performed on 25 µg of protein extracts from various
mouse tissues using antibodies against PSTPIP and PTP-PEST.
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Little is known concerning the expression pattern of PSTPIP at the
protein level. Before investigating the interactions between PTP-PEST
and PSTPIP, we examined if the proteins were significantly co-expressed
in a panel of mouse tissues. As seen in Fig. 1C, PSTPIP was
detected in high amounts in spleen and thymus, followed by a more
moderate expression in lung, brain, and muscle lysates. Low levels of
PSTPIP were detected in heart and liver and no protein was detected in
kidney extracts. We do not know at the present if the faster migrating
band observed in heart and liver lysates corresponds to a PSTPIP-like
protein or a degradation product. PTP-PEST expression significantly
overlaps with the pattern of PSTPIP at the protein level (Fig.
1C). In particular, the two proteins are highly present in
spleen, thymus, lung, and brain and both proteins are absent from
kidney lysates. Because PSTPIP was most highly found in spleen and
thymus, we compared the expression of PTP-PEST and PSTPIP in a panel of
hematopoietic cells. PSTPIP was highly expressed in two pro-B cell
lines, 70Z/3 and BaF/3, and in RAW macrophages. Lower levels of PSTPIP
were found in two T-cell lines, Jurkat and H9 (not shown). In this set
of cell lines, PTP-PEST was detected at high level in all samples. In
conclusion, the patterns of expression of PSTPIP and PTP-PEST are very
similar and this suggests that the study of their interaction might be of biological significance.
PSTPIP Is Associated with PTP-PEST in Fibroblasts and Hematopoietic
Cells--
We investigated if PSTPIP interacts with PTP-PEST in
vivo by a co-immunoprecipitation experiment. BaF/3 pro-B cells and
NIH 3T3 fibroblasts were lysed and PSTPIP was immunoprecipitated with an anti-PSTPIP antibody. Blotting with a polyclonal antibody against PTP-PEST (number 2530) was performed to monitor the presence of the
co-precipitated PTP-PEST. As seen in Fig.
2A, PTP-PEST was detected in
PSTPIP immunoprecipitates both from BaF/3 and NIH 3T3 (top
panel, lanes 2 and 4). As a control, no
PTP-PEST was found when the preimmune serum was used (lanes
1 and 3). The blot was reprobed with an anti-PSTPIP-HRP
antibody to verify the precipitation of PSTPIP (bottom
panel). In addition, we also observed the association between the
two proteins when PTP-PEST was immunoprecipitated (not shown). Thus,
PSTPIP and PTP-PEST are found together in a complex in vivo
both in fibroblasts and hematopoietic cells.
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Fig. 2.
PSTPIP interacts with PTP-PEST in
vivo. A, PSTPIP was immunoprecipitated from
the lysates of BaF/3 and NIH 3T3 cells and the coprecipitation of
PTP-PEST was monitored (top panel). The precipitation of
PSTPIP was verifed by reprobing the blot with an antibody against
PSTPIP. Neither PSTPIP nor PTP-PEST was detected in preimmune cell
lysates. B, the CTH domain of PTP-PEST is required for the
binding of PSTPIP. Wild type, Pro1, Pro2, and CTH PTP-PEST
were transiently co-transfected with PSTPIP in HEK293-T cells. The
expression of PSTPIP in each lysates was verified by immunoblotting
with the antibody against PSTPIP (top panel). PTP-PEST was
immunoprecipitated and the binding of PSTPIP was analyzed using the
antibody against PSTPIP (middle panel). No PSTPIP was
co-precipitated with CTH PTP-PEST. The precipitation of PTP-PEST
from each lysate was verified by reprobing the blot with a polyclonal
antibody against PTP-PEST (bottom panel). C, the
CTH domain of PTP-PEST interacts with PSTPIP in vitro.
PSTPIP was in vitro transcribed/in vitro
translated in the presence of [35S]methionine and
subjected to an in vitro binding assay to either GST alone
of GST PTP-PEST CTH domain. D, tryptophan 232 in the
coiled-coil domain of PSTPIP is required for binding to PTP-PEST. Wild
type, W232A, Y344F, Y367F PSTPIP were co-transfected along with
PTP-PEST in HEK293T cells. Equal amounts of cell lysates were
immunoprecipitated with an antibody against PSTPIP and the binding of
PTP-PEST was analyzed by Western blotting (middle panel).
The W232A mutation completely abolished the interaction between PSTPIP
and PTP-PEST. Equal amounts of PSTPIP were immunoprecipitated as seen
by reprobing the blot with a polyclonal antibody against PSTPIP
(bottom panel). PTP-PEST expression was verified by
immunoblotting 10 µg of the lysates with an antibody against PTP-PEST
(upper panel). E, HA-PTP-PEST was expressed in
293-T cells and tested for its ability to interact with GST, GST PSTPIP
SH3, or GST PSTPIP full-length. A Coomassie Blue-stained gel of the
fusion protein used in this experiment is shown to demonstrate the
integrity of the proteins.
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The CTH Domain of PTP-PEST Is Required to Bind PSTPIP--
We next
wanted to characterize the requirement for the association between
PTP-PEST and PSTPIP. The carboxyl end of PTP-HSCF is highly homologous
to PTP-PEST carboxyl terminus. This region of PTP-HSCF was termed the
CTH domain and was shown to be involved in the binding of PSTPIP. The
association between PTP-PEST and PSTPIP was reconstituted in a 293-T
cell system. We did not detect endogenous PTP-PEST and PSTPIP in this
cell line with our antibodies. PTP-PEST constructs (WT, Pro1,
Pro2, and CTH) were transiently co-transfected with PSTPIP.
PTP-PEST was immunoprecipitated from each lysate (including mock and
PSTPIP alone controls) and the co-precipitation of PSTPIP was analyzed
by Western blotting with the anti-PSTPIP-HRP antibody. As observed in
Fig. 2B (middle panel), PSTPIP was precipitated
with WT, Pro1, and Pro2 PTP-PEST but was completely absent from a
precipitation of PTP-PEST CTH. The precipitation of the various
PTP-PEST proteins was verified by Western blotting (Fig. 2B,
bottom panel). The expression of PSTPIP was ascertained by
Western blotting the total cell lysate with the anti-PSTPIP antibody
(Fig. 2B, top panel). In support of these data,
we observed that the isolated CTH domain of PTP-PEST expressed as a GST
fusion protein was sufficient to interact with
[35S]PSTPIP in a pull-down assay as seen in Fig.
2C. We conclude that the PTP-PEST CTH domain is responsible
for its direct interaction with PSTPIP.
The Coiled-coil Domain of PSTPIP Is Responsible for the Interaction
with PTP-PEST--
To investigate, in vivo, the functional
domain of PSTPIP involved in the binding of PTP-PEST, we reconstituted
the association between PTP-PEST and PSTPIP (WT, W232A, Y344F, and
Y367F) in 293-T cells. We mutated a critical tryptophan residue (W232A)
in the PSTPIP coiled-coil domain known to be essential for the binding to PTP-HSCF (43). As shown in Fig. 2D (middle
panel), PTP-PEST co-precipitated with WT, Y344A, and Y367A PSTPIP
but not with PSTPIP W232A. The blot was stripped and reprobed with an
anti-PSTPIP-HRP antibody (Fig. 2D, bottom panel).
The expression level of PTP-PEST was assayed by Western blot analysis
of the lysates (Fig. 2D, top panel). Furthermore, we
observed that a GST fusion of the SH3 domain of PSTPIP was not able to
precipitate PTP-PEST. However, the GST fusion of the full-length PSTPIP
did interact strongly with HA-PTP-PEST suggesting the involvement of
the amino terminus of PSTPIP in this process as seen in Fig.
2E.
PTP-PEST and PSTPIP Co-localize in COS-1 Cells--
To support our
biochemical interaction data, we investigated if PTP-PEST and PSTPIP
could co-localize in COS-1 cells by immunofluorescence microscopy. The
cells were transiently transfected with plasmids encoding PSTPIP and
enhanced green fluorescent protein (EGFP)-tagged PTP-PEST (WT or
CTH). As seen in Fig. 3, A
and B, the PTP-PEST CTH mutant was detected as a diffuse
signal throughout the cytoplasm and the co-transfected PSTPIP gave a
fibrous staining and was also detected at the cortex of the stained
cells. The two proteins were not significantly co-localized as observed
by analyzing the yellow signal in the images overlay (Fig.
3C). PTP-PEST WT was also detected as a diffuse signal
throughout the cytoplasm but, interestingly, the co-expressed PSTPIP
was not detected as a fibrous signal but rather gave a diffuse
cytoplasmic staining in these conditions (Fig. 3, D and
E). Thus, there is a clear difference in the localization of
PSTPIP when it is expressed with either PTP-PEST WT or CTH. As
observed in the images overlay (Fig. 3F), almost all of the
PTP-PEST and PSTPIP were co-localized in the cytoplasm and the
perinuclear region as judged by the yellow signal. We verified that the
large GFP-tag added to PTP-PEST did not interfere with the ability of
PTP-PEST to associate with PSTPIP. As seen in panel G, GFP
PTP-PEST WT, but not CTH mutant, nicely co-precipitated with PSTPIP.
In summary, we found that PTP-PEST and PSTPIP are co-localized in COS-1
cells and that the expression of WT but not CTH PTP-PEST results in
a lost of the cortical and fibrous staining of PSTPIP.
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Fig. 3.
PTP-PEST and PSTPIP co-localize in COS-1
cells. COS-1 cells were transfected with PSTPIP in
combination with either EGFP PTP-PEST CTH (A and
B) or EGFP PTP-PEST wild-type (D and
E). The EGFP fusion proteins signal is found in panels
A and D while the TRITC signal (PSTPIP) is shown in
panels B and E. Panels C and
F correspond to the overlay of the EGFP and TRITC signals.
All fields were photographed using a ×40 Nikon objective.
G, EGFP PTP-PEST associates with PSTPIP. PSTPIP was
co-transfected with either EGFP PTP-PEST WT or CTH. PSTPIP was
immunoprecipitated and the bound PTP-PEST was detected by Western
blotting. The precipitation of PSTPIP was verified by reprobing with
the PSTPIP antibody. The expression of each proteins was also verified
by immunoblotting the cell lysates.
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PSTPIP Is Tyrosine Phosphorylated Upon PDGF and EGF Treatments in
NIH 3T3 Fibroblasts--
The pathways involved in inducing PSTPIP
tyrosine phosphorylation in vivo have not been fully
studied. In a previous report, activation of the PDGF receptor resulted
in the tyrosine phosphorylation of PSTPIP (15). This event was
dependent on c-Abl since in Abl / fibroblasts PSTPIP failed to
become tyrosine phosphorylated when the cells were treated with PDGF.
We investigated whether EGF, PDGF, and insulin could affect PSTPIP
tyrosine phosphorylation in mouse fibroblasts. NIH 3T3 and NIH 3T3
expressing the human EGF receptor (HER14) cells were serum starved and
treated with media alone, 25 ng/ml PDGF, 1 µM insulin or
pervanadate for 15 min. HER14 cells were stimulated with 100 ng/ml EGF
for 10 min. Each cell lysate was subjected to either a preimmune or an
anti-PSTPIP immunoprecipitation. As observed in Fig.
4A, treatment of NIH 3T3 cells
with PDGF results in a 2.4-fold increase in PSTPIP tyrosine phosphorylation levels (as determined by densitometry analysis) while
insulin had no effect (1.2-fold increase). Treatment of HER14 cells
with 100 ng/ml EGF stimulated the tyrosine phosphorylation of PSTPIP by
2.3-fold when compared with the serum-starved control. Finally,
treatments of HER14 cell with pervanadate lead to a dramatic increase
(4.5-fold) in PSTPIP tyrosine phosphorylation. The blot was reprobed
with the anti-PSTPIP-HRP to ascertained PSTPIP precipitation (Fig.
4A, lower panel).
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Fig. 4.
PSTPIP is tyrosine phosphorylated in
vivo. A, PSTPIP tyrosine phosphorylation
levels are increase following PDGF and EGF treatments of NIH 3T3
fibroblasts. NIH 3T3 and HER14 cells were serum starved and treated
either with the media, 25 ng/ml PDGF, 1 mM insulin, 100 ng/ml EGF or with pervanadate. Cells were lysed and PSTPIP was
immunoprecipitated and analyzed by anti-phosphotyrosine antibody
immunoblotting. The blot was reprobed with the anti-PSTPIP-HRP to
verify the precipitation of PSTPIP (bottom panel).
B, PSTPIP is tyrosine phosphorylated by various tyrosine
kinases in vivo. HEK293T cells were transiently transfected
with PSTPIP and plasmids encoding for various tyrosine kinases: v-Src,
c-Src, Fyn, Lck, Lyn, c-Abl, FAK, or Mock (empty pcDNA3.1 Zeo).
PSTPIP was immunoprecipitated and the levels of tyrosine
phosphorylation were analyzed by immunoblotting with an
anti-phosphotyrosine antibody (4G10-HRP). The precipitation of PSTPIP
was verified by reprobing the blot with an antibody against PSTPIP
(bottom panel). The expression of the tyrosine kinases was
verified by immunoblotting the cell lysates with the appropriate
antibodies. C, Src kinases are not involved in the
EGF-mediated phosphorylation of PSTPIP. HER14 cells were either treated
with DMSO or 10 µM PP2 prior to EGF stimulation. PSTPIP
was immunoprecipitated and analyzed by anti-phosphotyrosine Western
blotting (top panel). The blot was reprobed with antibodies
against PTP-PEST (middle panel) and PSTPIP (bottom
panel). The efficacy of the PP2 treatment was verified by
immunoblotting the total cell lysate (25 µg of proteins) with an
anti-phosphotyrosine antibody (D) and also by evaluating the
amount of c-Cbl precipitated by a GST Fyn SH2 fusion protein
(E).
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We screened a panel of tyrosine kinases involved in PDGF and EGF
receptor signaling to obtain insight on the mechanism involved in
PSTPIP phosphorylation. We investigated if members of the Src kinases
(v-Src, c-Src, Fyn, Lck, and Lyn), c-Abl or FAK are individually able
to phosphorylate PSTPIP following transient co-expression in 293-T
cells. PSTPIP was immunoprecipitated and its phosphotyrosine levels
were analyzed. As observed in Fig. 4B (top
panel), PSTPIP was tyrosine phosphorylated when co-transfected
with v-Src, c-Src, Fyn, Lyn, and c-Abl. The hematopoietic restricted
tyrosine kinase Lck and the focal adhesion kinase (FAK) were not able
to phosphorylate PSTPIP in this assay. The amount of precipitated
PSTPIP was evaluated by Western blotting with the anti-PSTPIP-HRP
antibody as seen in Fig. 4B (middle panel). With
the exception of Lck, we verified the expression of the tyrosine
kinases by Western blotting (bottom panel). In conclusion,
two widely distributed members of the Src family, c-Src and Fyn, and
c-Abl are candidates to mediate the PDGFR and EGFR dependent increase
in PSTPIP tyrosine phosphorylation in adherent cells. Furthermore, Lyn
could be responsible for PSTPIP phosphorylation in hematopoietic cells
such as B-lymphocytes and mast cells.
We next used the PP2 inhibitor to determine if Src kinases are involved
in the tyrosine phosphorylation of PSTPIP downstream of the EGF
receptor. The pretreatment of HER14 cells with PP2 did not block the
EGF-induced tyrosine phosphorylation of PSTPIP as shown in Fig.
4C (top panel). The blot was reprobed with the anti-PSTPIP-HRP antibody to verify equal precipitation (Fig.
4C, bottom panel). We also observed that EGF
stimulation does not affect the interaction between PTP-PEST and PSTPIP
(Fig. 4C, middle panel). The effect on the PP2
inhibitor was ascertained by analyzing the total tyrosine
phosphorylation in the cell lysate (Fig. 4D) and also by
looking at the amount of c-Cbl, a known Src substrate, that was
precipitated by the GST Fyn SH2 domain (Fig. 4E). In both
control experiments, we indeed observed that the PP2 inhibitor blocked
the activity of the Src kinases. We conclude that Src kinases are not
involved in the phosphorylation of PSTPIP downstream of the EGF
receptor and this result together with previous results (15) points to
c-Abl as the critical kinase involved in PSTPIP phosphorylation
downstream of growth factor receptors.
PTP-PEST Dephosphorylates Tyrosine 344 of PSTPIP and Prevents Its
Interaction with SH2 Domain-containing Proteins--
We next
investigated if PSTPIP is a substrate of PTP-PEST and if the binding
between the two proteins is required for an efficient dephosphorylation. PSTPIP and c-Abl were co-transfected with PTP-PEST WT, PTP-PEST CTH, PTP-PEST C231S, or PTP-PEST Pro3-5 C231S. The
various PTP-PEST proteins were immunoprecipitated with an antibody
recognizing PTP-PEST and all its mutants (serum number 2528). As
observed in Fig. 5A (top
panel), tyrosine-phosphorylated PSTPIP was precipitated by the
C231S mutant of PTP-PEST. The blot was reprobed with an antibody
against PSTPIP to demonstrate that the phosphorylated protein of 50 kDa
is PSTPIP (middle panel). PSTPIP precipitated with WT
PTP-PEST (lane 5) was dephosphorylated demonstrating that
PSTPIP is a substrate for the catalytic activity of PTP-PEST. When
c-Abl was not co-transfected, no tyrosine-phosphorylated PSTPIP could
be precipitated by either PTP-PEST WT or C231S (lane 1 and
3). To establish if the CTH domain of PTP-PEST plays a role in the substrate recognition, we used a substrate trapping mutant (C231S) with a large deletion of the PSTPIP-binding site (Pro3-5). As observed in Fig. 5A (top panel, lane
8), this mutant of PTP-PEST failed to precipitate any
phosphorylated protein of 50 kDa, clearly demonstrating that the
catalytic domain of PTP-PEST is not capable of interacting with
tyrosine-phosphorylated PSTPIP on its own. All the PTP-PEST constructs
in which the CTH domain is lacking did not interact with PSTPIP
(middle panel). The expression of PTP-PEST proteins was
verified by immunoblotting the total cell lysates with a polyclonal
antibody against PTP-PEST (bottom panel).
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Fig. 5.
Binding of the CTH domain of PTP-PEST to
PSTPIP is essential for the dephosphorylation of PSTPIP Y344.
A, HEK293T were transiently co-transfected with PSTPIP and
either PTP-PEST WT, WT CTH, C231S, or C231S Pro3-5 in the
presence or not of c-Abl to induce PSTPIP phosphorylation. PTP-PEST was
immunoprecipitated from the extracts with a polyclonal antibody. The
tyrosine phosphorylation levels of the co-precipitated PSTPIP were
verified using the anti-phosphotyrosine antibody 4G10HRP (top
panel). The presence of co-precipitated PSTPIP was detected by
Western blotting with an antibody against PSTPIP (middle
panel). Expression of PTP-PEST in the lysates was verified by
reprobing the blot with an antibody against PTP-PEST (bottom
panel). B, the same experiment as in A was
repeated with PSTPIP mutants Y344F and Y367F. C, PSTPIP
interacts with SH2 domains in vitro. The SH2 domains of Crk,
Fyn, c-Abl, Nck, and c-Src expressed as GST fusion proteins were tested for their ability to interact with
tyrosine-phosphorylated PSTPIP. Abl was co-transfected with WT, Y344F,
or Y367F PSTPIP and the lysates were subjected to a binding assay using
various GST SH2 domains. D, PTP-PEST regulates the binding
between PSTPIP and SH2 domain containing proteins by dephosphorylating
tyrosine 344. PSTPIP Y367F and c-Abl were co-transfected with or
without PTP-PEST. Each cell lysate was subjected to a precipitation by
GST alone, GST SH2 Abl or GST SH2 Fyn. The presence of the precipitated
PSTPIP was monitored by immunoblotting with the anti-PSTPIP-HRP
antibody. The presence and integrity of each fusion protein was
verified by Coomassie Blue staining of the membrane.
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We used tyrosine to phenylalanine mutants of PSTPIP (Y344F and Y367F)
to investigate which tyrosine(s) of PSTPIP are dephosphorylated by
PTP-PEST. c-Abl, PSTPIP, mutants, and PTP-PEST (WT or C231S) were
co-transfected in 293-T cells and PTP-PEST was immunoprecipitated from
each sample using the anti-PTP-PEST antibody (number 2530). As shown in
Fig. 5B (top panel), PSTPIP Y344F was not
detected in a tyrosine-phosphorylated form when co-immunoprecipitated
with PTP-PEST C231S trapping mutant. However, PSTPIP Y367F was detected as a highly tyrosine-phosphorylated protein in the PTP-PEST C231S precipitate (lanes 2 and 6) but was almost
completely dephosphorylated in the PTP-PEST WT precipitate (lanes
1 and 5). The blot was reprobed with an anti-PSTPIP
antibody to verify that PSTPIP and the mutants were co-precipitated
with the PTP-PEST proteins (Fig. 5B, middle panel). The lysates were immunoblotted with an anti-PTP-PEST
antibody to monitor the expression of PTP-PEST (bottom
panel). These results indicate that tyrosine 344 of PSTPIP is a
target of PTP-PEST catalytic activity. No conclusion could be made
about the action of PTP-PEST on the tyrosine 367 of PSTPIP in this
assay since this tyrosine failed to become tyrosine phosphorylated when
PSTPIP Y344F mutant was used with c-Abl or any other kinase that was
tested. This issue is addressed in Fig. 7 of this article. From these
data, we conclude that PTP-PEST dephosphorylates tyrosine 344 of
PSTPIP.
We investigated if the phosphotyrosines of PSTPIP could serve as
docking sites for SH2 domain containing proteins. To verify that
hypothesis, we transfected 293-T cells with PSTPIP in combination with
c-Abl to induce its tyrosine phosphorylation. In addition, since two
putative tyrosine phosphorylation sites are reported (19), the PSTPIP
mutants Y344F and Y367F were also co-transfected with c-Abl. Equal
amounts of the cell lysates were subjected to pull-down assays with the
SH2 domains of Crk, Fyn, c-Abl, Nck, or c-Src expressed as GST fusion
proteins. As observed in Fig. 5C, the SH2 domains of c-Src,
Fyn, and Abl precipitated PSTPIP. In contrast, the SH2 domain of Crk
precipitated a very small amount of PSTPIP. GST Nck SH2 domain and GST
alone did not precipitate PSTPIP. All the SH2 domains and GST alone did
not precipitate PSTPIP when c-Abl was not co-transfected (not shown).
We investigated if the SH2 domains of Fyn, c-Src, and c-Abl interacted
preferentially with any of the two putative phosphotyrosines of PSTPIP
(Tyr344 and Tyr367). PSTPIP Y344F was unable to
interact with any of the SH2 domain tested (Fig. 5C,
right panel). In fact, PSTPIP phosphorylation is completely
lost when tyrosine 344 is mutated (19). PSTPIP Y367F was effectively
precipitated by the same SH2 domains as the wild type protein, namely
the SH2 domains of Fyn, c-Abl, and c-Src. In addition, the amount of
PSTPIP Y367F precipitated by the SH2 domains was comparable with the
amount of wild type PSTPIP precipitated by the same SH2 domains
suggesting that Tyr344 is the major site involved in these interactions.
Having established that tyrosine 344 is a binding site for SH2
domain-containing proteins, we investigated if PTP-PEST could regulate
these associations by dephosphorylating tyrosine 344 of PSTPIP. PSTPIP
Y367F and c-Abl were co-transfected with or without PTP-PEST. Each cell
lysate was subjected to a precipitation with GST alone, GST SH2 c-Abl,
or GST SH2 Fyn. As presented in Fig. 5D, the SH2 domain of
c-Abl and Fyn precipitated a significant amount of PSTPIP Y367F but
this binding was lost when PTP-PEST was co-expressed (top
panel). The integrity of the GST fusion was verified by Coomassie
staining. The expression of PTP-PEST and PSTPIP Y367F was ascertained
by immunoblotting the lysates with the appropriate antibodies. In
summary, PTP-PEST is able to dephosphorylate PSTPIP at tyrosine 344 and
this event negatively regulates the association of PSTPIP with SH2
domain-containing proteins.
The Formation of the PSTPIP-WASP Complex Is Not Regulated by
Tyrosine Phosphorylation in Vivo--
It was previously reported that
the phosphorylation of tyrosine 367, which lies within the SH3 domain
of PSTPIP, negatively regulates the binding of PSTPIP with WASP
in vitro (19). We tested if this negative regulation also
occurs in vivo. WASP and PSTPIP were co-transfected with the
tyrosine kinases c-Abl and Fyn, and we subsequently verified the
stability of the PSTPIP·WASP complex. Surprisingly, even if
PSTPIP tyrosine phosphorylation levels were dramatically increased
under these conditions (Fig. 4B and data not shown), the
formation of the PSTPIP·WASP complex was not affected as observed in
Fig. 6A (middle
panel). The expression of WASP was detected in each lysate and
similar amount of PSTPIP were immunoprecipitated as shown in the
upper and lower panel of Fig. 6A.
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Fig. 6.
The PSTPIP·WASP complex is stable in
vivo but can be disrupted by v-Src. A, PSTPIP and
Flag-tagged WASP were co-transfected with an empty vector, c-Abl, or
Fyn encoding plasmids. The level of Flag WASP in each transfection was
verified by blotting the lysates with an antibody against the Flag
epitope (upper panel). PSTPIP was immunoprecipitated and the
co-precipitation of WASP was assayed by blotting with the anti-Flag
antibody (middle panel). The immunoprecipitation of PSTPIP
was verified by reprobing the blot with an anti-PSTPIP antibody
(bottom panel). B, in a similar assay, v-Src
ability to interfere with the PSTPIP·WASP complex was tested. PSTPIP
and WASP were transfected with v-Src and/or PTP-PEST. PSTPIP was
immunoprecipitated and Western blotting with the anti-Flag antibody
monitored in the presence of co-precipitated WASP. The blot was
sequentially reprobed with an anti-4G10 and an anti-PSTPIP antibody.
The expression of WASP and PTP-PEST was also verified by immunoblotting
the lysates with the anti-Flag and anti-PTP-PEST antibodies.
C, the dissociation of PSTPIP and WASP mediated by v-Src is
independent of PSTPIP tyrosine phosphorylation. PSTPIP Y367F mutant was
transfected with either an empty vector or with v-Src. PSTPIP Y367F was
immunoprecipitated and the presence of WASP was assayed by blotting
with the anti-Flag antibody.
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It was originally reported that v-Src can induce the
phosphorylation of tyrosine 367 of PSTPIP, at least by monitoring the formation of a slower migrating form of PSTPIP by SDS-PAGE (15, 19). We
investigated if this viral form of Src is capable of abrogating the
PSTPIP·WASP complex in vivo. As observed in Fig. 6B, WASP does not co-immunoprecipitate with PSTPIP when
v-Src is co-expressed (lanes 1 and 2). The
co-expression of PTP-PEST was not sufficient to restore the binding
between PSTPIP and WASP under these conditions (lane 3).
Surprisingly, the co-expression of PTP-PEST only resulted in a 2-fold
decrease in the phosphorylation of PSTPIP (middle panel).
Interestingly, we observed that in the samples transfected with v-Src,
the migration of PTP-PEST on SDS-PAGE was significantly retarded
(lane 3, PTP-PEST blot). We failed to detect tyrosine
phosphorylation of PTP-PEST under these conditions to explain this
slower migration in SDS-PAGE. It is an intriguing possibility that
v-Src expression could affect PTP-PEST catalytic activity. It is
possible that a v-Src-activated serine kinase phosphorylates and
down-regulates PTP-PEST catalytic activity since serine phosphorylation
was proposed to regulate PTP-PEST activity (44). To demonstrate if the
loss of association between PSTPIP and WASP results from the
phosphorylation of tyrosine 367, we repeated the experiment using
PSTPIP Y367F mutant. As observed in Fig. 6C, v-Src was
unexpectedly able to prevent the association of PSTPIP and WASP even in
the absence of Tyr367 in PSTPIP. These results suggest that
the ability of v-Src to regulate the association of PSTPIP and WASP is
not dependent on the phosphorylation of tyrosine 367 of PSTPIP.
PSTPIP Is Not Phosphorylated on Tyrosine 367--
Experimental
evidence, such as retarded migration in SDS-PAGE and loss of WASP
binding in vitro, have led to the conclusion that PSTPIP can
be phosphorylated on tyrosine 367 (19). Furthermore, a model has
emerged suggesting that the phosphorylation of PSTPIP on tyrosine 367 is done via a sequential mechanism involving the prior recruitment of a
SH2 domain-containing tyrosine kinase to tyrosine 344 of PSTPIP (19).
In this article, we indeed provide some evidence that SH2
domain-containing tyrosine kinases can interact with PSTPIP in
vitro. However, our in vivo co-immunoprecipitation assays suggest either that phosphorylation of PSTPIP on tyrosine 367 is
not negatively affecting the interaction with WASP or that tyrosine 367 is simply not phosphorylated in vivo. To resolve this issue,
we performed tryptic phosphopeptide mapping of PSTPIP. HEK293-T cells
were transfected with PSTPIP or its Y344F and Y367F mutants together
with either c-Abl or v-Src. The cells were labeled in vivo
with 32P and PSTPIP was immunoprecipitated, transferred to
PVDF, and digested with trypsin. The peptides were recovered and
separated by electrophoresis and TLC. As seen in Fig.
7A, two major peptides of
PSTPIP (Y1 and Y2) are phosphorylated by c-Abl. Both of these peptides
are not phosphorylated in the PSTPIP Y344F mutant co-expressed with
c-Abl (data not shown). Interestingly, the Y1 and Y2 peptide are still
being phosphorylated by c-Abl in the PSTPIP Y367F mutant (Fig.
7B). We obtained similar results when v-Src was transfected (Fig. 7, C and D). Y2 (c-Abl map) and Y3 (v-Src
map) do not migrate identically on TLC. By deduction, Y1 must include
tyrosine 344. Importantly, we conclude that PSTPIP is not
phosphorylated on tyrosine 367 in vivo. However, we do not
exclude the possibility that other tyrosine residues (Y2 and Y3) in
PSTPIP are also phosphorylated. The phosphorylation of these tyrosine
residues in PSTPIP would have to be dependent on the prior
phosphorylation of tyrosine 344.
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Fig. 7.
PSTPIP is not phosphorylated on tyrosine 367 in vivo. PSTPIP WT, Y344F, and Y367F were
co-transfected with either c-Abl (A and B) or
v-Src (C and D). The cells were labeled with
[32P]orthophosphate and PSTPIP was immunoprecipitated
from each condition, purified by SDS-PAGE, and subjected to a tryptic
digest. The peptides were separated in two dimensions by
electrophoresis followed by thin layer chromatography. The
phosphopeptides were detected by autoradiography. As observed
previously, the Y344F mutant was not phosphorylated and was thus not
subjected to phosphopeptide mapping. As seen in A, two
phosphorylated peptides (Y1 and Y2) were detected when PSTPIP is
phosphorylated by c-Abl. None of these peptides were affected by the
mutation Y367F as observed in panel B suggesting that c-Abl
does not phosphorylate PSTPIP on tyrosine 367. Similar results were
obtained when v-Src was used as a kinase (Y1 and Y3, panels
C and D).
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PSTPIP Serves as a Scaffold Guiding PTP-PEST for a Specific
Dephosphorylation of WASP--
WASP has been shown to become tyrosine
phosphorylated upon adhesion of platelets to collagen, engagement of
the B cell-receptor in B-lymphocyte, and IgE cross-linking in mast
cells (45-48). WASP is phosphorylated on tyrosine 291 mainly by two
tyrosine kinases in these cells: Lyn and Btk (47, 48). The biological
function of this tyrosine phosphorylation of WASP has not yet been
elucidated. However, recent data on the inactive versus
active structure of WASP have provided interesting insight on a
putative function of this tyrosine phosphorylation (49). WASP is known
to be in an inactive conformation at the resting state. This dormant
state of WASP is mediated by an intramolecular binding involving its GTPase-binding domain and the VCA tail. In such a conformation, WASP is
unable to interact with the Arp2/3 complex because of its buried VCA
tail. This stands as an important mechanism for the regulation of actin
nucleation (28). Upon interaction of the inactive WASP with GTP-bound
CDC42 via the GTPase-binding domain, WASP undergoes a dramatic
conformational change and the VCA domain is exposed and can now
interact with the Arp2/3 complex and promote actin nucleation.
Interestingly, when WASP is in its inactive conformation,
Tyr291 is buried in an helix and not readily available
for tyrosine phosphorylation. Upon binding of CDC42 and conformational
rearrangements, Tyr291 is now accessible to tyrosine
kinases because of the disruption of the helix. Moreover, it has
been proposed that phosphorylation of Tyr291 could
destabilize the inactive conformation of WASP and thus help to keep
WASP in an active state (28). In this model, an important aspect of the
down-regulation of WASP activity would be the dephosphorylation of
Tyr291 in order for the protein to fold into an inactive
conformation in which the VCA is masked in the intramolecular interaction.
We tested if PTP-PEST can dephosphorylate WASP on Tyr291.
PTP-PEST is unable to interact directly with WASP. Since PSTPIP
interacts strongly with both PTP-PEST and WASP, we investigated if it
could act as a scaffold between these two molecules. As seen if Fig. 8A, WASP was detected in a
PTP-PEST immunoprecipitation. No WASP was detected when the preimmune
serum was used. The bridging molecule PSTPIP was also present in the
complex as expected (middle panel). The precipitation of
PTP-PEST was verified by Western blotting (bottom panel).
Flag-WASP becomes tyrosine phosphorylated when the Lyn tyrosine kinase
is co-expressed as observed in Fig. 8 (lane 1) and in a
previous report (47). We found that PTP-PEST can partially
dephosphorylate WASP when PSTPIP is co-transfected (lane 2).
However, if a PSTPIP mutant (W232A) unable to interact with PTP-PEST is
transfected, WASP is significantly less dephosphorylated by PTP-PEST
(lane 3). Furthermore, if a PTP-PEST C231S is transfected, WASP becomes slightly more tyrosine phosphorylated suggesting that the
PTP-PEST mutant can interact with and protect WASP in a
tyrosine-phosphorylated state. The immunoprecipitation of WASP was
verified by reblotting with the anti-Flag antibody (middle panel). Furthermore, the expression of PSTPIP and PTP-PEST
proteins was ascertained by Western blotting the cell lysates with the appropriate antibodies. Together, these data suggest that WASP is a
substrate of PTP-PEST and that the specificity of this biological event
is mediated by the scaffolding action of the adapter protein PSTPIP.
This result could have important implications in the regulation of
WASP-like proteins activity toward the Arp2/3 complex and actin polymerization.
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Fig. 8.
PSTPIP act as a scaffold guiding PTP-PEST for
a specific dephosphorylation of WASP. A, PTP-PEST was
immunoprecipitated from RAW macrophage lysate. The co-precipitation of
WASP and PSTPIP was analyzed by Western blotting. The preimmune
antiserum was used as a control. B, Lyn has been previously
described as a kinase capable of phosphorylating WASP on tyrosine 291. In conditions in which WASP, Lyn, and PSTPIP were co-expressed, WASP
was detected in a tyrosine-phosphorylated state. When PTP-PEST was
transfected, a significant decrease in WASP tyrosine phosphorylation
was noted but this effect of PTP-PEST could be counteracted by the
transfection of the PSTPIP W232A mutant, which is unable to interact
with PTP-PEST. WASP phosphorylation was slightly increased in the
presence of the C231S mutant of PTP-PEST.
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DISCUSSION |
We previously noted the hyperphosphorylation of PSTPIP in
fibroblasts lacking PTP-PEST suggesting that it might be a substrate for this enzyme (13). In this article, we provide evidence that PTP-PEST and PSTPIP physically interact and that this binding is
critical for a specific dephosphorylation of PSTPIP on
Tyr344. Furthermore, we show that PSTPIP is phosphorylated
downstream of the activated PDGF and EGF receptors. This
phosphorylation of PSTPIP is most likely mediated by c-Abl since we
show that the Src kinases are not involved in this process. We
performed phosphopetides maps to clearly demonstrate that PSTPIP is not phosphorylated on Tyr367. This is an important finding
since it suggests that the association between PSTPIP and WASP would
not be regulated by the phosphorylation of this residue in
vivo. Interestingly, we show that PSTPIP can act as a linker
between PTP-PEST and WASP leading to a specific dephosphorylation of WASP.
The analysis of the expression of the mRNA of PSTPIP by Northern
blotting suggests an enrichment in lymphoid organs (18). However,
PTP-PEST is known to be ubiquitously expressed (4, 6, 40). We studied
the distribution of PSTPIP at the protein level and we found that it is
widely expressed in several murine tissues (spleen, thymus, brain,
lung, and muscle) and hematopoietic cells (pro-B cells, macrophages and
less abundantly in T-cells). We also detected PSTPIP in NIH 3T3
fibroblasts. The expression of another PTP known to target PSTPIP,
PTP-HSCF, is restricted to hematopoietic stem cells (18, 50). These
protein expression patterns clearly suggest that a more widely
expressed PTP must replace PTP-HSCF in other cell types to regulate the
phosphorylation of PSTPIP, we propose that PTP-PEST is this candidate.
Our data suggests that PTP-PEST and PSTPIP patterns of expression
overlap significantly and that the study of their interactions is of
biological significance.
Endogenous PSTPIP and PTP-PEST could be co-precipitated from NIH 3T3
fibroblasts and 70Z/3 pro-B cells suggesting that these proteins are
included in the same complexes in vivo. We further demonstrated that the carboxyl-terminal homology (CTH) domain of
PTP-PEST is required to mediate the association with PSTPIP both in a
co-precipitation experiment and in an in vitro binding assay. We conclude that the CTH domain of PTP-PEST is necessary for the
binding to PSTPIP. Interestingly, PSTPIP was originally cloned as a
PTP-HSCF interacting protein and the CTH domain of PTP-HSCF was shown
to be responsible for the interaction (18). Since the CTH domain of
PTP-PEST is 75% identical to the one of PTP-HSCF, our results are in
agreement with an observation by Spencer et al. (18) that a
PTP-PEST peptide containing the CTH domain can disrupt the association
between PSTPIP and PTP-HSCF. Conversely, our results demonstrate that
the coiled-coil domain of PSTPIP is involved in mediating the
interaction with PTP-PEST both in vivo and in
vitro. A mutation of a tryptophan residue in the coiled-coil
domain of PSTPIP (W232A) disrupts the association between PSTPIP and
PTP-PEST. This same mutant of PSTPIP is also impaired in its ability to
interact with PTP-HSCF, but it still localizes properly with the actin
cytoskeleton suggesting no major disruption of the protein folding
(43). Hence, the interactions between PTP-PEST and PSTPIP are conserved
at the molecular level when compared with the association of PSTPIP
with PTP-HSCF. It is expected that the other PEST-like PTPases PEP and
BDP will also bind PSTPIP in an identical manner.
To support our data on the physical association between PTP-PEST and
PSTPIP, we found that both proteins co-localize in COS-1 cells by
immunofluorescence analysis. This co-localization was significantly
reduced when the CTH deletion mutant of PTP-PEST was studied. A
surprising observation from those experiments is that the cellular
localization of PSTPIP is dramatically affected whether it is bound or
not to PTP-PEST. We found that PSTPIP is present at the cortical actin
when co-expressed with PTP-PEST CTH. However, most of the PSTPIP is
found in the cytoplasm and the perinuclear region when co-transfected
with the WT PTP-PEST. This effect of PTP-PEST on the PSTPIP
localization could be a mechanism that regulates the function of PSTPIP
by promoting its turnover at the cortical actin. Interestingly, this
result also suggests that in future studies the CTH domain of PTP-PEST
could be used as a dominant-negative mutant that would prevent the
adequate localization of PSTPIP.
During this study, we observed that PSTPIP is not significantly
tyrosine phosphorylated in proliferating NIH 3T3 fibroblasts. We found
that PDGF and EGF receptor activation can result in an increase in
PSTPIP tyrosine phosphorylation. Both PDGF and EGF receptors
stimulation lead to the activation of several downstream kinases. In
both pathways, the Src family of kinases, c-Abl and FAK are known to
play important roles (51-53). We found that c-Abl, c-Src, and Fyn are
competent to phosphorylate PSTPIP in a transient transfection assay.
However, the PP2 inhibitor did not block the EGF-induced
phosphorylation of PSTPIP strongly suggesting that the Src kinases are
not involved in the phosphorylation of PSTPIP downstream of the EGF
receptor. During the completion of this work, Cong et al.
(15) found that PSTPIP becomes tyrosine phosphorylated in mouse
fibroblasts following PDGF treatment. This phosphorylation was mainly
under the control of c-Abl since PSTPIP failed to become tyrosine
phosphorylated in Abl / fibroblasts (15). To support the hypothesis
that c-Abl can directly phosphorylate PSTPIP, it was shown that the SH3
domain of PSTPIP directly interacts with two proline-rich sequences of
c-Abl and that this interaction increases the specificity of the kinase
reaction (15). From these data, c-Abl is probably the critical tyrosine
kinase downstream of the PDGF and EGF receptors involved in PSTPIP
tyrosine phosphorylation.
It has been reported that two major sites of tyrosine phosphorylation
exist on PSTPIP: Tyr344 and Tyr367 (19).
Indeed, it was demonstrated that PSTPIP is phosphorylated on tyrosine
344 in cells treated with the PTP inhibitor pervanadate or cells
transfected with v-Src since a point mutation of this amino acid to a
phenylalanine completely prevented PSTPIP tyrosine phosphorylation
(19). However, phosphorylation of PSTPIP on tyrosine 367 was proposed
based on the observation that when Y367F mutant was subjected to
SDS-PAGE, a slower migrating band of PSTPIP was not detected in Western
blotting with an anti-phosphotyrosine antibody (19). We did not detect
any differences between the tyrosine phosphorylation levels of PSTPIP
WT and Y367F when these proteins were co-expressed with tyrosine
kinases. To clarify these findings, we performed an extensive analysis
of PSTPIP tyrosine phosphorylation. We found that PSTPIP is not
phosphorylated on tyrosine 367 (see Fig. 7). We conclude that the major
site of phosphorylation in PSTPIP is Tyr344. However, we
observed two phosphopeptides for PSTPIP in our maps. We do not rule out
the possibility that other tyrosine residues could be phosphorylated in
PSTPIP. However, these other phosphorylation sites would have to be
dependent on the prior phosphorylation of Tyr344. This
hypothesis is currently being
tested.2
Tyrosine 344 of PSTPIP is found in a YASI motif. We tested several SH2
domains for their ability to bind phosphorylated PSTPIP and we found
that the SH2 domains of c-Abl, and of the Src kinases c-Src and Fyn are
capable of such a function in vitro. We also show that
mutation of tyrosine 367 of PSTPIP does not affect the binding of the
SH2 domain of the tyrosine kinases to PSTPIP, but that mutation of
Tyr344 completely prevented the association with these SH2
domains. These results suggest that Tyr344 is the binding
site for these SH2 domains. The analysis of the Tyr344
motif, YASI, suggests that it does share some similarities with known
c-Src and Fyn SH2 domain-binding motifs. For example, the SH2 domains
of c-Src and Fyn can strongly interact with a YAEI motif found in FAK
(54, 55) suggesting that a YAxI motif could be sufficient for mediating
the association of these SH2 with PSTPIP. The biological functions of
the complexes formed between PSTPIP and SH2 domain-containing tyrosine
kinases may be to transmit the signals from activated EGF and PDGF
receptor. However, we failed to detect Src in PSTPIP immunoprecipitate.
Thus, further studies are required to determine if PSTPIP can interact
with SH2 domain-containing proteins in vivo.
Having established that PSTPIP becomes tyrosine phosphorylated in
vivo following growth factor stimulation, we tested if PSTPIP can
be a substrate for the catalytic activity of PTP-PEST. We found that
Tyr344 of PSTPIP is a specific site targeted by PTP-PEST.
Furthermore, we determined that the binding of PSTPIP to the PTP-PEST
CTH domain is essential for an efficient dephosphorylation of PSTPIP.
This result indicates that the catalytic domain of PTP-PEST possesses no intrinsic specificity toward phosphorylated PSTPIP, and that PTP-PEST requires another interaction for this enzyme-substrate recognition. This is in contrast to PTP-PEST recognition of
tyrosine-phosphorylated p130Cas in which the PTP domain of
PTP-PEST demonstrates a marked specificity and is sufficient to
recognize p130Cas (3, 5).
The interaction between PSTPIP and WASP was proposed to be regulated by
the phosphorylation of tyrosine 367 located within the SH3 domain of
PSTPIP (56). However, this observation could not be reproduced under
in vivo situations and we also failed to detect
phosphorylation of Tyr367 in vivo. In this
respect, the stability of the PSTPIP·WASP complex in vivo
was not affected by the co-transfection of wild-type tyrosine kinases
such as c-Abl and Fyn. We conclude that it is unlikely that the
association of PSTPIP and WASP will be regulated by tyrosine phosphorylation in vivo. Nonetheless, we found that the
PSTPIP-WASP association is completely abolished in v-Src expressing
cells and that this event is independent of Tyr367
phosphorylation. The mechanism by which v-Src mediate this effect on
the PSTPIP·WASP complex is not understood at the moment.
A major interest from the present work is the potential modulation of
WASP tyrosine phosphorylation by PTP-PEST. In cells, WASP and N-WASP
interaction with the Arp2/3 is tightly regulated by an autoinhibition
mechanism of the WASP proteins (28, 29). In fact, when native WASP is
purified to homogeneity from bovine thymus, it behaves poorly to
activate actin nucleation via the Arp2/3 complex (31). Several
independent studies have shown that WASP proteins are maintained
inactive by intramolecular interactions involving the WA domain and the
GTPase-binding domain of these proteins. In this autoinhibited state,
the Arp2/3-binding region is hidden and WASP proteins are not capable
of promoting actin nucleation. The best understood mechanism for the
activation of WASP proteins involves the simultaneous interaction of
the proteins with GTP-loaded Cdc42 and the phospholipid
PIP2 (33). These interactions lead to the disruption of the
intramolecular interaction and to the exposition of the Arp2/3-binding
site of the WASP proteins. Several other mechanisms of activation of
these proteins have also been reported involving SH3 domain-containing
proteins and other interacting protein such as WIP but are not
understood in great details (34-36, 57). The possibility of expressing
and purifying full-length WASP and N-WASP or their separate domains from bacteria, and using these proteins for in vitro actin
nucleation assays in the presence of the purified Arp2/3 complex have
been extremely useful to study the activation mechanisms of the WASP proteins. However, is this intramolecular interaction the only way of
regulating WASP proteins? Another mechanism of regulation that has not
been studied carefully is the tyrosine phosphorylation of WASP
proteins. WASP is phosphorylated on tyrosine 291 following a variety of
stimuli such as interaction of platelets with collagen, the engagement
of the B-cell receptor in B-lymphocytes and the IgE receptor in mast
cells (45-48). Two tyrosine kinases, Btk and Lyn, are reported to be
able of phosphorylating WASP. The phosphorylation of N-WASP has not yet
been studied. The biological consequences of the tyrosine
phosphorylation of WASP remain completely unknown. However, a recent
structural analysis of WASP has provided an interesting hypothesis
regarding WASP phosphorylation. In this report, the three-dimensional
structure of WASP was elucidated by NMR spectroscopy and provided the
first structural evidence of the WASP autoinhibited and activated
conformations. Interestingly, tyrosine 291 resides in the helix 3 in the inhibited conformation of WASP. According to the structural
data, this tyrosine residue is buried inside the helix and is not
likely to be accessible to tyrosine kinases. However, when WASP is
activated, tyrosine 291 is available for phosphorylation by tyrosine
kinases. In agreement with this model, the co-expression of activated
Cdc42 with WASP facilitates WASP tyrosine phosphorylation by Lyn (47).
Kim et al. (49) proposed that the phosphorylation of
tyrosine 291 could stabilize WASP in the active conformation by
preventing the refolding of helix 3 which is present in the inhibited
conformation (49). This could be an important mechanism to prolong the
duration of WASP signaling in vivo. It will be important to
investigate if recombinant WASP fragments phosphorylated on tyrosine
291 behave differently in in vitro actin nucleation assay.
We found that PSTPIP can form a trimolecular complex with PTP-PEST and
WASP. Since PSTPIP can interact both with WASP and PTP-PEST, we tested
if WASP is a substrate of PTP-PEST. We found that PTP-PEST can
dephosphorylate WASP. Furthermore, we uncovered that the scaffolding
role o
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ABSTRACT
INTRODUCTION
