Physical and Functional Interactions between Receptor-like Protein-tyrosine Phosphatase alpha  and p59fyn*

Vijay Bhandari, Kah Leong Lim, and Catherine J. PallenDagger

From the Cell Regulation Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have examined the in vivo activity of receptor-like protein-tyrosine phosphatase alpha  (PTPalpha ) toward p59fyn, a widely expressed Src family kinase. In a coexpression system, PTPalpha effected a dose-dependent tyrosine dephosphorylation and activation of p59fyn, where maximal dephosphorylation correlated with a 5-fold increase in kinase activity. PTPalpha expression resulted in increased accessibility of the p59fyn SH2 domain, consistent with a PTPalpha -mediated dephosphorylation of the regulatory C-terminal tyrosine residue of p59fyn. No p59fyn dephosphorylation was observed with an enzymatically inactive mutant form of PTPalpha or with another receptor-like PTP, CD45. Many enzyme-linked receptors are complexed with their substrates, and we examined whether PTPalpha and p59fyn underwent association. Reciprocal immunoprecipitations and assays detected p59fyn and an appropriate kinase activity in PTPalpha immunoprecipitates and PTPalpha and PTP activity in p59fyn immunoprecipitates. No association between CD45 and p59fyn was detected in similar experiments. The PTPalpha -mediated activation of p59fyn is not prerequisite for association since wild-type and inactive mutant PTPalpha bound equally well to p59fyn. Endogenous PTPalpha and p59fyn were also found in association in mouse brain. Together, these results demonstrate a physical and functional interaction of PTPalpha and p59fyn that may be of importance in PTPalpha -initiated signaling events.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 PTPalpha , a receptor-type PTP.

PTPalpha 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 PTPalpha 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 PTPalpha may normally play a role in stimulating cell proliferation. The intracellular mediators of PTPalpha signaling are not known. The tyrosine kinase pp60c-src is a candidate PTPalpha substrate since PTPalpha overexpression in rat embryo fibroblasts and P19 cells results in pp60c-src dephosphorylation and activation (20, 21). PTPalpha may also exert some of its cellular effects through its ability to bind the adaptor protein Grb2 (24-27). Downstream components in a PTPalpha signaling pathway may include mitogen-activated protein kinase and the transcription factor c-Jun, both of which are activated in PTPalpha -overexpressing rat embryo fibroblast cells (28). Whether PTPalpha 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 PTPalpha substrates. The identification of PTPalpha substrates is an important step in elucidating the biological role of PTPalpha . In this study, we have investigated the action of PTPalpha 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 PTPalpha (1). In addition, PTPalpha is highly expressed in brain (18, 29), and PTPalpha , pp60c-src, and p59fyn are implicated in or associated with certain neuronal cell functions including neuronal differentiation (PTPalpha (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 PTPalpha -directed cellular events.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression Plasmids-- Numbering of the PTPalpha amino acid sequence is according to Krueger et al. (17). The expression vector pXJ41-PTPalpha -neo, encoding full-length PTPalpha , has been described (20). The mutagenesis altering the essential cysteine residues to serine residues in the active sites of PTPalpha (C414S/C704S) has been described (39), and a fragment of this cDNA was used to replace the corresponding piece of wild-type PTPalpha cDNA to produce pXJ41-PTPalpha (C414S/C704S)-neo. Vectors expressing VSVG-tagged versions of PTPalpha 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 PTPalpha cDNAs of pXJ41-neo, permitting the expression of PTPalpha 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 lambda 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 PTPalpha (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 PTPalpha (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-PTPalpha antiserum 3897 (raised against a glutathione S-transferase/PTPalpha -D2 fusion protein (39) containing the second catalytic domain and C-terminal tail region of PTPalpha ) 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-PTPalpha 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-PTPalpha (3680, raised against a peptide comprising the C-terminal 18 amino acids of PTPalpha ), anti-p59fyn (FYN3, Santa Cruz Biotechnology, Inc.), or anti-Csk (C-20, Santa Cruz Biotechnology, Inc.) polyclonal antibodies. In some experiments, anti-PTPalpha antibody 3680 was blocked before immunoprecipitation by preincubation with recombinant purified PTPalpha -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-PTPalpha 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 PTPalpha 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 [gamma -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

PTPalpha Effects Dephosphorylation of pp60c-src and p59fyn-- The ability of PTPalpha 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 PTPalpha 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 PTPalpha (data not shown). As p59fyn represents a novel potential substrate for PTPalpha , we characterized the catalytic action of PTPalpha toward p59fyn in more detail. The extent of p59fyn dephosphorylation increased as increasing amounts of PTPalpha cDNA were transfected, reaching a plateau of 80-90% tyrosine dephosphorylation (Fig. 2A). Dephosphorylation was completely dependent on the catalytic activity of PTPalpha since a PTPalpha mutant, PTPalpha (C414S/C704S), in which the essential cysteine residues in the active sites of both catalytic domains of PTPalpha were mutated to serine residues, was unable to effect p59fyn dephosphorylation (Fig. 2A).


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Fig. 1.   Coexpression of PTPalpha with p59fyn and p60c-src results in dephosphorylation of p59fyn and p60c-src. COS-1 cells were transfected with empty plasmid (mock), 3 µg each of p59fyn and pp60c-src cDNAs together, or 3 µg each of p59fyn, pp60c-src, and PTPalpha cDNAs together, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. Immunoprecipitates of p59fyn (A) or pp60c-src (B) from 250 µg of the cell lysates were probed with anti-phosphotyrosine (top panels) and anti-p59fyn (A) or pp60c-src (B) (bottom panels) antibodies. HC, heavy chain antibody.


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Fig. 2.   Dose-dependent dephosphorylation and activation of p59fyn by PTPalpha . A, lysates of COS-1 cells transfected with 2 µg of p59fyn cDNA alone or together with the indicated amounts of wild-type PTPalpha or the double catalytic mutant PTPalpha (C414C/C704S) cDNA were resolved by SDS-polyacrylamide gel electrophoresis and probed with anti-phosphotyrosine antibody, and the signal was quantitated by densitometry. The p59fyn dephosphorylation in the presence of PTPalpha (closed squares) and PTPalpha (C414S/C704S) (open squares) was calculated by taking the phosphotyrosine content of p59fyn from COS-1 cells expressing p59fyn alone as 100%. Points shown are the means from three independent experiments, and the error bars indicate the mean ± S.E. B, COS-1 cells were transfected with 2 µg of p59fyn cDNA alone (lane 1) or with increasing amounts of wild-type PTPalpha cDNA (lane 2, 0.5 µg; lane 3, 1 µg; lane 4, 2 µg; lane 5, 4 µg; lane 6, 6 µg). p59fyn was immunoprecipitated from 500 µg of lysate, and a portion was used in an immunocomplex kinase assay (top panel) while the rest was probed for p59fyn (middle panel). Cell lysates were probed for PTPalpha (bottom panel). The results shown are from one experiment, but are representative of the results of three independent experiments.

Coexpression of PTPalpha and p59fyn Results in p59fyn Activation-- Besides tyrosine dephosphorylation of p59fyn, the coexpression of PTPalpha resulted in kinase activation of p59fyn. As shown in Fig. 2B, when p59fyn immunoprecipitates from cells coexpressing PTPalpha were used in an immunocomplex kinase assay, increasing p59fyn autophosphorylation was observed with increasing expression of PTPalpha . A maximum 5-fold increase in kinase activity was obtained (Fig. 2B, lanes 5 and 6) at the same PTPalpha /p59fyn cDNA ratio observed to give maximal p59fyn dephosphorylation (Fig. 2A). No increase in the kinase activity of p59fyn was measured when catalytically inactive PTPalpha 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 PTPalpha 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, PTPalpha -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 PTPalpha /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 PTPalpha -mediated dephosphorylation of the C-terminal Tyr-531 of p59fyn.


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Fig. 3.   Accessibility of the p59fyn SH2 domain is increased upon coexpression with PTPalpha . COS-1 cells were transfected with 4 µg of p59fyn cDNA alone or together with 4 µg of PTPalpha cDNA as indicated, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. Lysates of cells expressing p59fyn in the absence (lane 1) or presence (lane 2) of PTPalpha were immunoblotted with anti-PTPalpha (A), anti-p59fyn (B), and anti-phosphotyrosine (C) antibodies, respectively. The cell lysates (300 µg) were incubated with unphosphorylated Src Tyr-527 peptide (D, top panel) or Src Tyr-527 phosphopeptide (bottom panel) coupled to Sepharose beads as described under "Experimental Procedures." The washed precipitates were immunoblotted with anti-p59fyn antibody.

Specificity of PTPalpha in Effecting p59fyn Dephosphorylation and Activation-- To examine whether the PTPalpha -mediated p59fyn dephosphorylation was a specific effect of PTPalpha 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 PTPalpha (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 PTPalpha - 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 PTPalpha -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 PTPalpha , but not CD45, correlated with elevated kinase activity of Fyn in PTPalpha -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 PTPalpha -mediated dephosphorylation and activation of p59fyn reflect a specific effect of PTPalpha .


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Fig. 4.   PTPalpha (but not CD45) expression induces dephosphorylation of p59fyn. A, cells were transfected with 4 µg of p59fyn cDNA and 4 µg of PTPalpha or CD45 cDNA, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. Membrane (M; lanes 1, 3, and 5) and cytosol (C; lanes 2, 4, and 6) fractions were prepared from lysates of COS-1 cells expressing p59fyn alone or together with PTPalpha or CD45 as indicated and immunoblotted with anti-p59fyn (top panel), anti-PTPalpha (middle panel), and anti-CD45 (bottom panel) antibodies. B, cell fractions (0.5 µg of protein) were assayed for phosphatase activity toward phosphotyrosyl-RR-Src peptide as described under "Experimental Procedures." Activity of the membrane fraction of the PTPalpha -expressing lysate was taken as 100%. C, immunoprecipitates of p59fyn from 500 µg of total cell lysates were probed for phosphotyrosine (top panel) and p59fyn (bottom panel).

Association of PTPalpha and p59fyn-- To further investigate the interaction of PTPalpha and p59fyn, we examined whether these two proteins underwent any form of association. To enable immunoprecipitation of PTPalpha , a 29-amino acid epitope from VSVG was inserted into the PTPalpha extracellular region to create VSVG-PTPalpha . Cells expressing p59fyn alone or in conjunction with VSVG-PTPalpha were lysed under mild detergent conditions, and PTPalpha was immunoprecipitated with anti-VSVG antibodies. Probing of the immunoprecipitates with anti-VSVG antibodies detected two forms of VSVG-PTPalpha : a broad diffuse band migrating at ~130 kDa, consistent with the size of wild-type glycosylated PTPalpha , and a sharper band at ~100 kDa, consistent with the size of incompletely glycosylated PTPalpha (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-PTPalpha (Fig. 5A, bottom panel, lanes and 4) and a significant amount of p59fyn in the anti-VSVG immunoprecipitate from the PTPalpha /p59fyn-coexpressing cells (lane 8). The presence of this p59fyn in the VSVG immunoprecipitate was due to its interaction with VSVG-PTPalpha 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-PTPalpha (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 PTPalpha and p59fyn were carried out with cell lysates prepared by solubilization with Brij 96, a mild non-ionic detergent. The association of PTPalpha 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 PTPalpha .


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Fig. 5.   Association of PTPalpha and p59fyn in vivo. A, COS-1 cells were transfected with empty plasmid (8 µg; mock), p59fyn cDNA (4 µg), VSVG-PTPalpha cDNA (4 µg), or VSVG-PTPalpha and p59fyn cDNAs (4 µg each) as indicated. The total amount of DNA in each transfection was equalized by the addition of empty plasmid. Total cell lysates (lanes 1-4) and VSVG-PTPalpha immunoprecipitates (IP) prepared from 400 µg of lysates (lanes 5-8) were immunoblotted with anti-VSVG (top panel) and anti-p59fyn (bottom panel) antibodies. A p59fyn immunoprecipitate from the cells expressing p59fyn alone was probed with anti-p59fyn antibody (lane 9). B, a portion of the p59fyn immunoprecipitate from p59fyn-expressing cells (lane 1) and portions of the VSVG-PTPalpha immunoprecipitates described above (lanes 2-5) were assayed for in vitro kinase activity as described under "Experimental Procedures." HC, heavy chain antibody.

Reciprocal experiments were carried out to confirm the association of PTPalpha and p59fyn in which p59fyn immunoprecipitates from transfected cells were analyzed for the presence of PTPalpha . Probing with anti-VSVG antibodies revealed the presence of VSVG-PTPalpha in p59fyn immunoprecipitates from PTPalpha /p59fyn-expressing cells (Fig. 6A, lane 6), but not in immunoprecipitates from p59fyn- or PTPalpha -expressing cells (lanes 4 and 5). Although two forms of VSVG-PTPalpha are present in whole cell lysates, likely corresponding to glycosylated and incompletely glycosylated forms of PTPalpha (50), only the larger glycosylated form was found in the p59fyn immunoprecipitates. It is possible that the less glycosylated PTPalpha is incompletely processed and trapped in the endoplasmic reticulum and/or Golgi apparatus, and its absence in the p59fyn immunocomplex suggests that PTPalpha and p59fyn interaction represents a specific cellular protein-protein association that does not merely occur as a post-lysis event. When PTPalpha was replaced with CD45 in the transfections, no co-immunoprecipitated CD45 was detected in p59fyn immunoprecipitates from CD45/p59fyn-expressing cells (Fig. 6B). The above results correlated with the presence of PTP activity when portions of the p59fyn immunoprecipitates analyzed in Fig. 6 (A and B) were assayed. Low levels of PTP activity were present in the p59fyn immunoprecipitates from cells transfected with p59fyn, PTPalpha , or CD45 alone or with p59fyn and CD45 together. A much higher level of PTP activity was present in the p59fyn immunoprecipitates from cells coexpressing p59fyn and PTPalpha (Fig. 6C). The same results were obtained when untagged PTPalpha was used in the experiment (data not shown), indicating that the VSVG tag does not interact with p59fyn. Thus, despite the higher PTP activity in lysates of CD45-expressing cells compared with PTPalpha -expressing cells (Fig. 6C, inset), only activity attributable to PTPalpha co-immunoprecipitated with p59fyn.


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Fig. 6.   PTPalpha (but not CD45) associates with p59fyn. Cells were transfected with 2 µg of p59fyn cDNA and 2 µg of PTPalpha cDNA or 4 µg of CD45 cDNA, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. A, whole cell lysates (lanes 1-3) and immunoprecipitates (IP) of p59fyn from 800 µg of lysates of COS-1 cells expressing p59fyn, VSVG-PTPalpha , or p59fyn and VSVG-PTPalpha (lanes 4-6) were probed with anti-VSVG (top panel) and anti-p59fyn (bottom panel) antibodies. B, whole cell lysates (lanes 1-3) and immunoprecipitates of p59fyn from 800 µg of lysates of COS-1 cells expressing p59fyn, CD45, or p59fyn and CD45 (lanes 4-6) were probed with anti-CD45 (top panel) and anti-p59fyn (bottom panel) antibodies. C, phosphatase assays of phosphotyrosyl-RR-Src peptide were performed with p59fyn immunoprecipitates from lysates of COS-1 cells expressing p59fyn (a, open diamonds), VSVG-PTPalpha (b, open squares), p59fyn and VSVG-PTPalpha (c, closed squares), CD45 (d, open triangles), or p59fyn and CD45 (e, open circles). The inset shows the RR-Src phosphatase activity of these cell lysates (104 cpm released per 3 min/µg of lysate) prior to immunoprecipitation.

Association of p59fyn and a Catalytic Mutant of PTPalpha -- The mechanism and temporal occurrence of PTPalpha and p59fyn association (pre- or post-dephosphorylation) were examined using a catalytically inactive form of PTPalpha . 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-PTPalpha 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 PTPalpha . However, if p59fyn dephosphorylation is prerequisite for PTPalpha -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-PTPalpha (C414S/C704S) (data not shown, but see Fig. 2A). Nevertheless, p59fyn was detected in immunoprecipitates of VSVG-PTPalpha (C414S/C704S) at a level equivalent to that of p59fyn in immunoprecipitates of catalytically active VSVG-PTPalpha (Fig. 7). Thus, a conventional substrate-trapping technique fails to enhance PTPalpha -p59fyn interaction, suggesting that additional or alternative regions of these proteins are responsible for association. Also, the finding that p59fyn and PTPalpha can associate independently of PTPalpha activity, and thus in the absence of or prior to dephosphorylation, indicates that PTPalpha is suitably positioned to directly utilize p59fyn as a substrate.


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Fig. 7.   Association of PTPalpha and p59fyn is independent of the catalytic state of PTPalpha . Anti-VSVG immunoprecipitates were prepared from COS-1 cells expressing p59fyn alone or together with either wild-type VSVG-PTPalpha or the double catalytic mutant (dm) VSVG-PTPalpha (C414S/C704S). Transfections used 4 µg of each cDNA, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. PTPalpha and p59fyn in cell lysates (lanes 1-3) and in the anti-VSVG immunoprecipitates (IP) prepared from 400 µg of cell lysates (lanes 4-6) were detected by immunoblotting with anti-VSVG (top panel) and anti-p59fyn (bottom panel) antibodies. HC, heavy chain antibody.

Association of Endogenous PTPalpha with p59fyn-- The above studies were carried out with COS cells ectopically expressing PTPalpha 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 PTPalpha is highly expressed in brain (18, 29) and because neuronal functions of both PTPalpha (21, 30) and p59fyn (34-36) have been reported. None of the PTPalpha antibodies available to us immunoprecipitated PTPalpha very efficiently, based on comparisons with the level of PTPalpha detected by Western blotting of brain lysates (data not shown). Some PTPalpha 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 PTPalpha , and specific immunoprecipitation of PTPalpha was blocked by preincubation of the antibody with recombinant purified protein (PTPalpha -D2) comprising the membrane distal catalytic domain and C terminus of PTPalpha (Fig. 8, lane 1). PTPalpha 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-PTPalpha antibody (3897) raised against recombinant PTPalpha -D2. Stripping and reprobing of lanes 1-4 with the anti-PTPalpha C-terminal antibody (3680, used for PTPalpha immunoprecipitations; see above) gave the same results (data not shown). This association of PTPalpha with p59fyn was observed in repeated experiments and demonstrates that these proteins are physiologically associated.


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Fig. 8.   PTPalpha associates with p59fyn in brain. Adult mouse brain lysate (1 mg) was incubated with a polyclonal antibody (3680) to the C terminus of PTPalpha , either directly (lane 2) or following preblocking of the antibody by prior incubation with recombinant PTPalpha antigen (PTPalpha -D2, which includes the C-terminal sequence of PTPalpha ) (lane 1), or was incubated with anti-p59fyn (lane 3) or anti-Csk (lane 4) polyclonal antibodies. The immunoprecipitates (IP) were probed with anti-PTPalpha antibody (3897) (upper panel) or anti-p59fyn antibody (lower panel). HC, heavy chain antibody.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have confirmed that PTPalpha dephosphorylates pp60c-src in vivo and found that PTPalpha 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 PTPalpha -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 PTPalpha on p59fyn. Previously, PTPalpha activity has mainly been assayed for artificial substrates in vitro using immunoprecipitated PTPalpha or bacterially expressed forms of cytosolic PTPalpha (24, 39, 56, 57). This characterization of a cellular assay of PTPalpha activity will be useful in the future assessment of the effects of various structural mutants of PTPalpha on its enzymatic function.

An important question regarding the activation of Src family kinases by PTPalpha is whether these kinases are directly dephosphorylated by PTPalpha and thus represent PTPalpha substrates. Potential PTPalpha 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 PTPalpha 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 PTPalpha did not associate with p59fyn. In addition, PTPalpha was detected in p59fyn immunoprecipitates from mouse brain. Although PTPalpha -p59fyn association is suggestive of a direct enzyme-substrate relationship, at present it is unclear whether these physical and functional actions of PTPalpha 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 PTPalpha to promote PTPalpha -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 PTPalpha with p59fyn indicates that this physical interaction does not require prior dephosphorylation/activation of p59fyn. We are generating truncated forms of PTPalpha 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 PTPalpha (RKYPPLP, amino acids 188-194). As PTPalpha is tyrosine-phosphorylated in the cell (25, 26), this could provide sites for SH2 binding. Alternatively, PTPalpha -p59fyn association may occur through other regions or intermediary proteins.

Besides the association with p59fyn described here, PTPalpha 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 PTPalpha 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 PTPalpha , but is not observed in the absence of the PTPalpha -Grb2(SH2) interaction. One effect of PTPalpha -Grb2(SH3) binding is postulated to be the inhibition of the catalytic activity of PTPalpha through the obstruction of substrate binding (26). Tyrosine phosphorylation of PTPalpha can be catalyzed by pp60c-src (24), and it will be of interest to see if p59fyn can phosphorylate PTPalpha at the same site to result in Grb2 binding. We have observed enhanced tyrosine phosphorylation of PTPalpha upon coexpression with p59fyn,2 although the site(s) of phosphorylation is unknown. If this phosphorylation occurs at the appropriate C-terminal site, PTPalpha -catalyzed activation of pp60c-src and/or p59fyn would activate kinase-mediated downstream signaling events while also permitting the feedback inhibition of PTPalpha 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 PTPalpha can occur on the same PTPalpha 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 PTPalpha expression may be mediated through pp60c-src (20, 21). Here we have provided further evidence that PTPalpha is a physiological regulator of the Src family kinases and, in particular, that p59fyn is a direct in vivo substrate of PTPalpha . 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 PTPalpha -expressing cells may be due to the increased kinase activity of p59fyn. Alternatively, PTPalpha -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 PTPalpha 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 PTPalpha 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, PTPalpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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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Discussion
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