May 17, 1994 - main in Grb2, as well as tyrosine phosphorylation of. R-PTP-a. This observation links a receptor tyrosine phosphatase with a key component of ...
THE JnmNAL OF B m m l c a CHEMISTRY Vol. 269,No. 29,Issue of July 22, pp. 18731-18734, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication Receptor Tyrosine Phosphatase R-PTP-aIS Tyrosine-phosphorylated and Associated with the Adaptor Protein GrbZ* (Received for publication, March 30, 1994, and in revised form, May 17, 1994)
Jing Su, Andreas Batzer, and Jan Sap* From the Department of Pharmacology, New York University Medical Center, New York, New York 10016
phorylated and associated withthe adaptor protein Grb2. The latter, composed entirely of one SH2 and two SH3 domains (141, is thought to provide coupling between (via its SH2 domain) tyrosine phosphorylation sites associated with activated tyrosine kinases or their substrates, and (via its SH3 domains) has downstream effectors. Genetic and biochemical evidence identified theSos protein, a guanine-nucleotide releasing factor for Ras, as a major effector for Grb2.Thus, Grb2 couples tyrosine kinase activation to Ras and its downstream kinase cascade (15,16).In addition, other Grb2-SH3 domain-binding proteins have been reported (17, 18). O u r evidence that Grb2 is also associated, via its SH2 domain, with R-PTP-a suggests that this adaptor molecule may also couple a R-PTPase tocomponents of the cellular signal transduction system.
Receptortyrosinephosphatases(R-PTPases)have EXPERIMENTALPROCEDURES generated interest because of their suspected involveCell Culture and Expression-Rat L6 myoblast and human embryoment in cellular signal transduction. The adaptor pro- nal kidney 293 cells were maintained in Dulbecco’smodifiedEagle’s tein Grb2 has been implicatedin coupling receptor ty- medium + 10% fetal calf serum. Stable lines (L6) were generated by rosinekinasesto Ras. We reportthat aubiquitous cotransfecting mouse R-PTP-a cDNA in pMJ30 with pSV2neo and by R-PTPase, R-FTP-a,is tyrosine-phosphorylated and as- G418 selection (19). Transient transfections (293 cells) were performed sociated in vivo with theGrb2 protein. This association using cytomegalovirus-basedvectors (20). The Nck and p85cDNAs can be reproducedin stably and transiently transfected were reported (21,22). Mouse Crk cDNA wasprovided byDr. A. cells, as well as in vitrousing recombinantGrb2 protein. Sorokin. Antisera and Protein Analysis-Anti-R-PTP-a antiserum 35, against Association requires the presence of an intactSH2 dothe R-PTP-a C terminus, was described (6); antiserum 210 was genermain in Grb2, as well as tyrosine phosphorylation of ated against a peptide corresponding t o aminoacids 509-523 of R-PTP-a.Thisobservationlinks areceptortyrosine R-PTP-a. For immunoblotting,these antisera were affinity-purifiedusphosphatase with a key component of a central cellular ing standard procedures.Anti-Grb2 antiserum 327was generated signaling pathway and provides a basis for addressing against the recombinant N-terminal SH3 domain of Grb2. Anti-Grb2 R-PTP-a function. antiserum 86 has been described elsewhere (141, as has themethod for generating anti-phosphotyrosineantiserum 72 (Ref. 23; provided by Dr. B. Margolis). Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology, Inc. Immunoprecipitations were for 90 min a t 4 “C in A large family of R-PTPases’ has been characterized (1, 2). Triton buffer (50 mM Hepes pH 7.5, 1%Triton X-100, 150 nm NaCl, 1.5 Although their transmembrane topology has prompted specu- m~ MgCl,, 1mM EGTA, 10%glycerol) with 1 nm Na,VO,, 50 m~ NaF, lation about their role in signal transduction, clues aboutthe 10 m~ Na,P,O,, 1mM phenylmethylsulfonyl fluoride, 10 pg/mlleupeptin, and 10 pg/ml aprotinin. Cells were growingin 10%fetal calf serum biological function of this class of enzymes are scarce. The before lysis. In the experiment in Fig. l B , 1 mM iodoacetic acid, 30 mM is requiredfortyrosinephosphorylation CD45R-PTPase events during activation of B- a n d T-lymphoid cells. This re- p-nitrophenyl phosphate, and 0.1 m~ phenylarsine oxide were used as additional PTPase inhibitors. In the experiment demonstrating the rea function of CD45 in dephosphorylation quirement for tyrosine phosphorylation of R-PTP-a (Fig. m),Na,VO, quirement may reflect of negative regulatory sites in the kinases Lck and Fyn (3). and Na,P,O, were omitted, and thelysates incubated with 400 units of Interestingly, CD45 itself contains a site of tyrosine phospho- YOP, PTPase (New England Biolabs) in the presence of 5 mM DTT and 2.5 mM EDTA for 30 min at 37 “C before the in vitro binding assay. rylation, which may regulate its catalytic activity. The SH2 Pervanadate treatment of cells (0.1 mM Na,VO, + 3 mM H,O,) was for 40 domain of t h e Lck kinase has been reported to bind to this min at 37 “C. Immunoprecipitates were washed five times with lysis phosphorylation site (4, 5). buffer (+ 1 m~ Na,VO,) and analyzed by immunoblotting using HRPThe R-PTPase R-PTP-a exhibits a widespread pattern of exprotein A or HRP-anti-mouse IgG (Amersham Corp.) and chemilumipression, suggesting an important role in cellular physiology nescence reagent (DuPont). (6-11). Overexpression of R-PTP-a has been reported to lead to Glutathione S-’Dunsferase Fusion Proteins and in Vitro Binding dephosphorylation of t h e T y r 5 2 7 residue in c-Src, and thus to Assays-Generation and purification of glutathione S-transferase fuincreased c-Srckinase activity (12, 13).However, fewd a t a pro- sion proteins corresponding to wild-type Grb2, Grb2-R86K, and Grb2of R-PTP-a at physiological levels P49L were described(14,24,25). 10 pg immobilized protein were incuvide insight into the function with 1 mgof lysate for 2 h at 4 “C, the complexes washed four of expression. We report here that R-FTP-a is tyrosine-phos- bated times with lysis buffer (+ 1 mM Na,VO,) and analyzed by SDS-PAGE and immunoblotting. * This work was supported by a Human Frontier Science Program RESULTS Organization long term fellowship(to J. Sap),American Cancer Society Institutional Grant IRG-14-35, and Sugen, Inc. The costsof publication Qrosine Phosphorylation ofR-PTP-a“L6 cells express a low of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent”in ac- level of endogenous R-PTP-a, accommodate efficient overexpression of R-FTPases, and have been used successfully to cordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence shouldbe addressed: Dept. of Pharmacol- study tyrosine phosphorylation events (19, 26). We generated ogy, NYU Medical Center, 550 First Ave., New York, NY 10016. Tel.: an R-PTP-a-overexpressing clone, a-12, by stable transfection 212-263-7120; Fax: 212-263-7133. The abbreviations used are: R-PTPase, receptor tyrosine phospha- with a mouse R-PTP-a cDNA expression vector. Immunoblottase; PTPase, protein tyrosine phosphatase; HRP, horseradish peroxi- ting of lysates with affinity-purified anti-R-PTP-a antibodies dase; PAGE, polyacrylamide gel electrophoresis; SH, Src homology. (Fig. lA)reveals the endogenous and transfectedR-PTP-a as
18731
Association of R-PTP-awith Grb2
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FIG.1. '&rosine phosphorylation of R-PTP-a. A, expression of R-PTP-a in parental L6 and transfected a-12 cells. Lysates were separated by SDS-PAGE andimmunoblottedwithaffmity-purifiedantiR-PTP-n antiserum 35. R, L6 and a-12 cells were subjected to immunoprecipitation with preimmune serum or anti-R-PTP-a antiserum 210, and the Precipitated proteins analyzed by immunoblotting with antiphosphotyrosine antibody 4G10.
FIG.2. Association ofR-FTP-a with Grbf. Cells weresubjected to immunoprecipitation with anti-Grb2 antiserum 327, and the immune complexes analyzed by blotting with anti-R-PTP-a antibody 35, antiphosphotyrosine antiserum 72, or anti-Grb2 antiserum 86.
130-kDa proteins, as described (6, 11).In addition, a 70-kDa protein was detected in a-12 cells with several anti-R-PTP-a antisera. Since i t is smaller than the R-PTP-a core protein, it may constitute a degradation or processing product of the 130kDa R-PTP-a protein. The different levels of R-PTP-a in both cells serve as a control in the subsequent experiments. The R-PTPase CD45 and two cytosolic PTPases have been reported to undergotyrosinephosphorylation (4, 5, 27-29). Anti-phosphotyrosine immunoblotting of total lysates revealed increased tyrosine phosphorylation of a130-kDa protein in a-12 cells as the only detectable difference with L6 cells (not shown), leading us to investigate whetherR-PTP-a was itself tyrosine-phosphorylated.Lysateswere prepared from L6 or a-12 cells and subjected to immunoprecipitationwith antiR-PTP-a antiserum. Immunoblottingof the precipitated complex with a monoclonal anti-phosphotyrosineantibody detected a 130-kDa protein in anti-R-PTP-a immunoprecipitates from L6 cells (Fig. 1B). This protein was not observed when using preimmuneserum for immunoprecipitation andwas more abundant in immunoprecipitatesfrom a-12 cells, confirming its identity as R-PTP-a. A low level of tyrosine phosphorylation was also detected in the 70-kDa R-PTP-a protein species in a-12 cells. Identical results were obtained using an antiserum against a different epitope in R-PTP-a for immunoprecipitation or using a different, polyclonal, anti-phosphotyrosineantiserum for immunoblotting (not shown). R-PTP-a Is Associated with Grh2 in Vivo-Tyrosine-phosphorylated residues can function a s docking sites for proteins containing SH2 domains,which recognize phosphotyrosine within the context of a specific amino acid sequence (16). preliminary A screening of SH2 domain containing proteins revealed that R-PTP-a could bind Grb2 in vitro (not shown). We subsequently asked whether both proteins were associated in vivo. Lysates from L6 or a-12 cells were subjected to immunoprecipitation with anti-Grb2 antiserum, and thewashed immune complexes were analyzed by blotting withanti-R-PTP-a antibodies. Fig. 2 shows that R-PTP-a protein was found in Grb2immunoprecipitates from L6 cells and was present a t higher levels in Grb2 immune complexes from the R-PTP-a-overexpressing clone a-12, although similar amounts of Grb2 wereprecipitated form each cell type. Anti-phosphotyrosine immunoblotting also reveals a 130-kDa tyrosine-phosphorylated protein, comigrating with R-PTP-a, in Grb2 immune complexes. Coimmunoprecipitation of Grb2 and R-PTP-a from L6 and a-12 cells was observed using different polyclonal or monoclonal anti-Grb2 antibodies for immunoprecipitation, and with three different antiR-PTP-a antisera for immunoblotting (notshown). Association of R-PTP-a and Grb2 Requires the Grb2 SH2 Domain-To investigate themechanism of R-PTP-dGrb2 association, we performed transient coexpression in 293 cells. An
expression vector for R-PTP-a was transfected together with vectors for wild-type or mutant forms of Grb2. The Grb2-R86K protein contains a mutation in the FLVR motif in its SH2 domain, which cripples its binding activity, while the Grb2-P49L mutation affects the function of the N-terminal SH3 domain (14, 30). Althoughequal amountsof the various Grb2 proteins wereexpressed and precipitated by anti-Grb2 antiserum in these experiments (not shown), the R-PTP-a protein was not associated with transfectedGrb2-R86K beyond the extent seen with endogenous 293 Grb2 protein. Introduction of the Grb2P49L mutation lead to partially reduced association compared to wild-type Grb2 (Fig. 3A ). Thus, since mutation of the FLVR motif, which is part of the conserved phosphotyrosine binding site of SH2 domains (31),abolishes association, Grb2/R-PTP-a association is likely to involve a canonical SH2lphosphotyrosine interaction. Tyrosine-phosphorylated residues in proteins, when associated with SH2 domains, are partially protected from phosphatase actions (32). Thus,if the Grb2-SH2 domain directlyinteracts with a phosphotyrosine residue in R-PTP-a, Grb2 overexpression would be expected to lead to hyperphosphorylation of R-PTP-a. To test thisprediction, a R-PTP-a expression vector was transfected into 293 cells, alone, or together with expression vectors for Grb2, Grb2R86K, or other SH2 domain containing proteins. To avoid possible artifacts due to dephosphorylation during immunoprecipitation, the transfected cells were lysed directly in hot SDS-PAGE sample buffer and the lysates separatedby SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting. This experiment (Fig. 3B) revealed the appearanceof a majornew tyrosine-phosphorylated protein of 130 kDa in cells transfected with R-PTP-a and wild-type Grb2 together. This protein migrated with a slightly higher mobility than a ubiquitous 140-kDa protein presentin all samples. Tyrosine phosphorylation of the 130-kDa protein was not observed in theabsence of cotransfected R-PTP-a, whenan R-PTP-a expression vector was cotransfectedin thepresence of the Grb2R86K mutant, or when vectors for several other SH2 domain containing proteins(Nck, Crk, and p85) were cotransfected. To confirm that the 130-kDa protein corresponded to tyrosine-phosphorylated R-PTP-a, this experiment was modified using, instead of wild-type R-PTP-a, a chimeric protein (molecular mass180 kDa) consisting of the extracellulardomain of the epidermalgrowth factor receptor fusedto the intracellular domain of R-PTP-a. In this case, a 180-kDa tyrosine-phosphorylated protein was detected upon cotransfection with wildtype Grb2 (Fig. 3C). In Vitro Association of Grb2 with R-PTP-a-We next wished to investigate whether complex formation between R-PTP-a and Grb2 could occur in vitro. To this purpose, immobilized
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FIG.3. R-PTF"dGrb2 association during transient coexpression. Expression vectors for R-PTP-a, X-B-1, wild-type Grb2, Grb2R86K, Grb2-P49L, Nck, Crk, or p85 were transiently transfected into 293 cells in various combinations.A, cells were lysed and subjected to anti-Grb2 immunoprecipitation,followed by anti-R-PTP-a immunoblotting. B , total lysates were prepared in SDS-PAGE sample buffer and subjected to anti-phosphotyrosine immunoblotting. C , a n experiment similar tothat inB was performed, using, instead of wild-type R-FTP-a, X-B-1 (a 180-kDa chimeric protein containingthe intracellular domain of R-FTP-a fused to the extracellular and transmembrane domains of the epidermal growth factor receptor). recombinant proteins corresponding t o wild-type or mutant Grb2 were allowed to equilibrate with lysatesfrom L6 or a-12 cells. After washing, the formed complexes were analyzed for the presence of R-PTP-a by immunoblotting. This experiment confirmed the in vivo results, in thatR-PTP-a protein present in the lysates bound to wild-type Grb2 and the Grb2-R86K mutation severely compromised binding. By contrast, theP49L mutation exerted only a marginal effect on complex formation (Fig. 4.4). Having an in vitroassay thatreflected the in vivobehavior of the complex, we tested the importanceof tyrosine phosphorylation of R-PTP-a for Grb2 binding. To this purpose, lysates were also prepared from a-12 cells pretreated with pervanadate to increase tyrosinephosphorylation of R-PTP-a or were prepared from the same cells in the absence of PTPase inhibitors and incubated with purified recombinant PTPase(33). Fig. 4B shows that R-FTP-aproteinisolated from pervanadatetreated cells displayed increased binding to recombinantGrb2 protein, whereas binding was abolished by PTPase treatment of the lysates. The effect of PTPase treatment of the lysates could be entirely inhibited by vanadate (data not shown). DISCUSSION
The simplest model fitting our observations involves a tyrosine-phosphorylated residue in R-PTP-a providing a binding site for the Grb2-SH2 domain. Atyrosine phosphorylation site in R-PTP-a has been mapped toT y P 'and has alsobeen shown to be required for Grb2IR-PTP-a association (34).The context of this site,YANF, is similar to otherGrb2-SH2 binding sites(24, 35). Whereas our data show that the Grb2-SH2 domain and tyrosine phosphorylation of R-PTP-a are required for complex formation, they do not allow the formal conclusion that this interaction is sufficient forcomplex formation, or direct. A mu-
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FIG.4. Zn vitro R-PTP-dGrb2 association. A, lysates from L6 or a-12 cells were incubated with immobilized recombinant glutathione S-transferase fusion proteinscorresponding to wild-type or mutant forms of Grb2, and the bound proteins analyzed by anti-R-PTP-a immunoblotting. B, a-12 cell lysates were prepared asA,inbut in addition were prepared from pervanadate-treated cells or prepared in the abYOP, PTPase before the in sence of PTPase inhibitors and treated with vitro binding assay.
tation in the SH3 domain of Grb2 appears to lead t o a small decrease inaffinity, and binding of R-PTP-a to the SH2 domain alone of Grb2 was less efficient than binding to full-length Grb2 (not shown). Whether thisreflects subtle interactions between the SH2 and SH3 domains, additional contacts between an SH3 domain of Grb2 and R-PTP-a, or the need for additional proteins to stabilize the complex, requires further study. The data have severalimplications for the understandingof R-PTPase function. First, by analogy with its role for receptor tyrosine kinases, Grb2 may allow R-PTP-a t o interact with downstream effectors or substrates, by mediating association between R-PTP-a and other proteinsor by guiding R-PTP-a to appropriate cellular locations (15,16,24,36). It is not clear yet whether R-PTP-a canrecruit a Grb2/Sos complex to the plasma membrane, thereby potentially modulating Ras function, or whether other proteins might be involved (15-18). Second, our data suggest an involvement of R-PTP-a, at physiological expression levels, in signal transductionprocesses. The ability of R-PTP-a to bind Grb2 may endow it with a function separate from its catalyticactivity. Thus, data thatR-PTP-a overexpression can induce transformation or differentiation by activating c-src may need to be viewed also in light of the function of Grb2 in Ras signaling (12-16). Third, the results raise possibility the of regulation of R-PTP-a function by tyrosine phosphorylation. Autodephosphorylation, possibly throughthegeneration of R-PTPase dimers induced by extracellular ligands, thus warrants investigation asa regulatory mechanism. Acknowledgments-We thank Drs. J. Schlessinger for stimulating the original stages of this work while J. Sap was in the Schlessinger laboratory; N. Li, W. Li, B. Margolis, 0. Silvennoinen, andA. Sorokin for reagents; and J. den Hertog andT.Hunter for early communication of mapping data on the tyrosine phosphorylation site in R-FTP-a. REFERENCES Charbonneau, H., and Tonks, N. K. (1992) Annu. Rev. Cell Biol. 8,463-493 Walton, K. M., and Dixon, J. E. (1993)Annu.Reu. Riochem. 62, 101-120 Weiss, A,, and Littman, D. R. (1994) Cell 76,263-274 Stover, D.,Charbonneau, H., Tonks, N. K., and Walsh. K.A. (1991)Proc.Natl. Acnd. Sei. U. S. A. 88,7704-7707 5. Autero, M., Saharinen, J., Pessa-Morikawa, T., Soula-Rothhut, M., Oetken, C., Gassmann, M., Rergman, M., Alitalo, K., Burn, P., Gahmberg, C. G., and 1. 2. 3. 4.
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