Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5002-5006, May 1994 Biochemistry --phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein PASCAL PERALDI*, ZHIZHUANG ZHAOt, CHANTAL FILLOUX*, EDMOND H. FISCHERt, AND EMMANUEL VAN OBBERGHEN** *Institut National de la Sante et de la Recherche M6dicale Unite 145, Facult6 de M6decine, 06107 Nice Cddex 2, France; and tDepartment of Biochemistry, University of Washington, Seattle, WA 98195 Contributed by Edmond H. Fischer, February 18, 1994

ABSTRACT Protein-tyroslne-phosphatase 2C (PTZC, PTP2C activity 5- to 10-fold (17). However, the precise role also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic of the association between the phosphatase and other mol- enzyme with two Src homology 2 dons. We have investi- ecules remains to be defined. Finally, in cells overexpressing gated its regulation by in PC12 rat phe both PTP2C and the EGF receptor, a marginal increase mocytoma cells. In untreated cells, PIP2C was phosphorylated (1.2-fold) in PTP2C activity can be induced by EGF (13). predominantly on residues. A 5-min treatment with In nonstimulated cells, PTP2C is phosphorylated mainly on epidermal growth factor (EGF) induced an increase in phos- serine and residues (15), whereas growth factors phorylation on threonine and, to a lesser degree, on serine. such as EGF and PDGF appear to lead to an increase mainly After 45 min of exposure to EGF, PTP2C phosphorylation in phosphotyrosine (13-15) with a smaller increment in returned to basal levels. Using an in vitro kinase assay, we phosphothreonine (14). The role of these multiple phospho- found that the 44-kDa mitogen-activated protein kinase, rylations of PTP2C has not been established. p44DIS,, phosphorylated PTP2C on serine and threonine res- sequence analysis has revealed that PTP2C contains several idues. This phosphorylation resulted in a pronounced inhibi- putative phosphorylation sites for the 44-kDa mitogen- tion of PTP2C enzyme activity measured with phorylated activated protein kinase (MAP kinase), p44mqP. The activity EGF receptors as substrate. Moreover, in intact PC12 cells, of this serine/threonine kinase depends on threonine and P7P2C was also inhibited following a short EGF treatment, but tyrosine phosphorylation regulated by an upstream kinase, its activity returned to normal when the exposure to EGF was MAP kinase/extracellular signal-regulated kinase (ERK) ki- maintained for 45 min. The profile ofthis response to EGF can nase (MEK) (18-21), and by phosphatase activities (22-25). be inversely correlated to that of the siulatory action ofEGF While the list of substrates for the MAP is continu- on p44-a. These data sugt that the EGF-induced regula- ously growing (26), the physiological targets for these en- tion of Pm C activity is meted by p44-i. These indis zymes are ill defined. provide evidence for an additi role ofthe ite-activated This study addresses the regulation ofPTP2C in PC12 cells. protein kinase cascade-namey, the regulation of a PTP. We show that, while PTP2C is phosphorylated mainly on serine residues in untreated cells, exposure to EGF leads to a Protein tyrosine phosphorylation has emerged as a key transient increase in threonine phosphorylation, the time mechanism for signal transduction leading to processes in- course of which follows the EGF-induced activation of volved in the control of an extraordinary variety of cellular p44m*. Further, p44maPk phosphorylates PTP2C on seine decisions affecting metabolism, proliferation, and differenti- and threonine residues in vitro. This phosphorylationresults in ation (1). The level oftyrosine phosphorylation is determined a decrease in PTP2C activity measured with the EGF receptor by the balance between the activities of protein-tyrosine as a substrate. The striking correlation between the effect of kinases and protein-tyrosine-phosphatases (PTPs) (2). Sche- EGF on the activity of p44mak and the effect on both the matically, PTPs fall into two groups, transmembrane and threonine/serine phosphorylation and the activity of PTP2C cytosolic enzymes (3, 4). Two known mammalian cytosolic suggests that PTP2C is negatively regulated by MAP kinase. PTPs possess Src homology 2 (SH2) domains, which allow them to associate with molecules phosphorylated on tyrosine MATERIALS AND METHODS residues. They are PTP1C (5), also known as SHPTP1 (6), Materials. Protein A-Sepharose CL4B, aprotinin, leupep- HCP (7), SHP (8), or PTPN6 (9), and PTP2C (10), also tin, Triton X-100, and EGF were purchased from Sigma. designated as SHPTP2 (11), SHPTP3 (12), orPTP1D (13); the Phenylmethylsulfonyl fluoride was from Serva, 32P, (28 Ci/ latter is related to the mouse Syp protein (14). While the pmol; 1 Ci = 37 GBq) from Amersham, and ['-t32P]ATP (7000 amino acid sequences ofPIP1C and PTP2C are homologous, Ci/mmol) from ICN. Polyclonal antibody against a peptide their tissue distributions differ. PTP1C is predominantly corresponding to sequence 984-996 ofthe EGF receptor was expressed in hematopoietic cells (6, 8), whereas PTP2C is a generous gift from J. Schlessinger (New York University). more widely distributed (10, 11). Antibody to PTP2C was raised in a rabbit against a SH2 Although the physiological function of PTP2C remains domain-truncated PTP2C expressed in Escherichia coli (9), unknown, recent studies have revealed the binding of the and antibody to MEK, against a 14-aa peptide derived from enzyme, through its SH2 domains, to tyrosine-phosphory- the N terminus of the protein. lated molecules such as the platelet-derived growth factor Buffers. Buffer A was 50 mM Hepes, pH 7.5/150 mM (PDGF) receptor (15), the epidermal growth factor (EGF) NaCl/10 mM EDTA/10 mM Na4P207/2 mM Na3VO4/100 receptor (13), and insulin receptor substrate 1 (16). Further, mM NaF/1% (vol/vol) Triton X-100. Buffer B was 50 mM a phosphotyrosine-containing peptide corresponding to the Hepes, pH 7.5/150 mM NaCl. Buffer C was 50 mM Hepes, binding site of PTP2C on the PDGF receptor increases the Abbreviations: PIP, protein-tyrosine-phosphatase; SH2, Src homol- The publication costs ofthis article were defrayed in part by page charge ogy 2; MAP kinase, mitogen-activated protein kinase; EGF, epider- payment. This article must therefore be hereby marked "advertisement" mal growth factor; PDGF, platelet-derived growth factor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5002 Downloaded by guest on September 24, 2021 Biochemistry: Peraldi et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5003 pH 7.0/50 mM NaCl. All the buffers were also supplemented Phosphorylation of EGF Receptors. PC12 cells were lysed with aprotinin (100 units/ml), leupeptin (20 mM), and phen- for 15 min in buffer B supplemented with 1% Triton X-100. ylmethylsulfonyl fluoride (0.18 mg/ml). Immunoprecipitates of EGF receptors were incubated at Cell Culture, Extrction, and Immunopecpltation. PC12 room temperature with 0.1 ,uM EGF in buffer B supple- cells were cultured in RPMI 1640 supplemented with 10%o mented with 0.1% Triton X-100 and 10%/ (vol/vol) glycerol. (vol/vol) horse serum, 5% (vol/vol) fetal bovine serum, After 20 min, 1 ,uM [y-32P]ATP (7000 Ci/mmol) and 5 mM penicillin (50 units/ml), and streptomycin sulfate (50 pg/ml). MnCl2 were added and the reaction mixture was further Prior to stimulation, cells were starved overnight in RPMI incubated for 1 min at 4°C. Thereafter, beads were washed 1640 containing 0.25% (vol/vol) fetal bovine serum and 0.2% twice with buffer C. (wt/vol) bovine serum albumin. After EGF stimulation, cells Phosphatase Asy. Immunoprecipitates of PTP2C were were washed with ice-cold phosphate-buffered saline and incubated at room temperature with the phosphorylated EGF then lysed in buffer A or B as described below. The lysates receptor in buffer C containing 8 mM 2-mercaptoethanol. were centrifuged at 18,000 x g for 30 min. Cell extracts were After 30 min, beads were washed with the same buffer and incubated for 1.5-2 hr with antibodies adsorbed on protein subjected to SDS/PAGE. A-Sepharose beads and then were washed twice with the SDS/PAGE and Phospho Amino Acid Analysis. SDS/ corresponding extraction buffers and twice with appropriate PAGE was run in a 7.5% polyacrylamide under reducing kinase or phosphatase assay buffers. conditions. For phospho amino acid analyses, sep- 32p Labeling of PC12 Cells. PC12 cells growing in 100-mm arated by SDS/PAGE were transferred to a poly(vinylidene dishes were washed twice in phosphate-free RPMI 1640 difluoride) membrane. Membrane segments containing medium and then incubated for 4 hr in the same medium PTP2C or the EGF receptor were excised, hydrolyzed, and supplemented with 32P1 (0.5 mCi/ml). Thereafter, 0.1 pM analyzed according to Kamps and Sefton (28). EGF was added and the cells were further incubated for S or 45 min. Proteins were solubilized in buffer A and immuno- RESULTS precipitated by antibody against PTP2C. PTPZC Is Phosphorylated in PC12 Cells. To investigate In Vitro Activation ofRecombinant p44I by Immunopre- whether EGF modifies the level of PTP2C phosphorylation, cipitatd MEK. Serum-starved PC12 cells were stimulated 32Pi-labeled PC12 cells were treated with EGF. After solu- with 0.1 juM EGF for 5 min. Cell extracts were made in buffer bilization, cell extracts were subjected to immunoprecipita- A. Immunoprecipitates of MEK were incubated with 4 pg of tion with antibody against PTP2C followed by SDS/PAGE recombinant rat p44maPk at 22°C (27) in 100 1d of 50 mM (Fig. 1 Left). While PTP2C showed basal phosphorylation in Hepes, pH 7.5/50 mM ATP/15 mM MgCl2/2 mM EGTA for untreated cells, the level of phosphorylation increased sig- 45 min. This treatment led to a >50-fold activation ofp44mPk nificantly after 5 min of EGF treatment. After exposure to (data not shown). Supernatant of this reaction mixture is EGF was maintained for 45 min, PTP2C phosphorylation referred to as activated p44mapk. returned to basal levels. As observed by other investigators In Vitro Phosphwylation of PTP2C by Activated p44mk. (14, 15), several phosphorylated proteins in the range 80-100 For immunopurification ofPTP2C, PC12 cells were sonicated kDacoprecipitated with the phosphatase. The nature ofthese for 10 sec in buffer B. Immunoprecipitates of PTP2C were proteins is unknown. incubated for 45 min at room temperature in 50 mM Hepes, Phospho amino acid analyses of PTP2C (Fig. 1 Right) pH 7.5/50 pM [y-_32P]ATP (400 Ci/mmol)/15 mM MgCl2/2 showed that, in untreated cells, PTP2C phosphorylation mM EGTA with or without activated p44fmaPk. occurred mainly on serine, with very little on threonine.

kDa P-SER [ * '0F. 206 - S P-THR [

105 - P-TYR [

70 - 4- PTP2C

43 -

Min of EGF Origin - treatment: 0 5 45 PTP2C from cells treated with EGF for: 0 5 45 min

FIG. 1. Phosphorylation of PTP2C in PC12 cells (Left) and phospho amino acid analysis (Right). Serum-starved PC12 cells were incubated with 32Pi for 4 hr and then with 0.1 IM EGF for 0, 5, and 45 min. Immunoprecipitates of PTP2C were analyzed by SDS/PAGE, and its phospho amino acid composition was assessed by high-voltage thin-layer chromatography. Representative autoradiograms ofthree different experiments with similar results are shown. P-SER, phosphoserine; P-THR, phosphothreonine; P-TYR, phosphotyrosine. Downloaded by guest on September 24, 2021 5004 Biochemistry: Peraldi et al. Proc. Natl. Acad. Sci. USA 91 (1994) (Fig. 2). Immunopurified PTP2C from unstimulated cells was Precipitalon cy PTP2C with: incubated in the presence of the kinase assay buffer alone 'a b P - (lane a) or supplemented with nonactivated (lane b) or c d activated (lane d) p44IaPk. A control incubation was per- formed under the same conditions as in lane d, except that kDa nonimmune antibodies were used in the immunoprecipitation P-SER- (lane c). No phosphoprotein was detected when PTP2C was 200 0 incubated in the absence of p44maPk or in the presence of P-THR - nonactivated p44mPk. In contrast, when PTP2C was incu- bated with activated p44mIPk (lane d), a majorphosphoprotein

116 - with a molecular mass identical to that ofPTP2C (68 kDa) was revealed. Note that no phosphoprotein was detected in the P-TYR - control incubation (lane c). Phospho amino acid analyses 97 - identified comparable phosphorylation on both serine and threonine residues (Fig. 2 Right). The fact that the 68-kDa _ 4 PTP2C phosphoprotein has a molecular mass compatible with that of 66 PTP2C and that it was seen only when antibody to PTP2C was used allowed us to recognize the 68-kDa phosphoprotein as PTP2C. This assignment is consistent with a similar phosphorylation by MAP kinase observed with recombinant PTP2C purified from an E. coli expression system (29). 45 - Phosphorylatton of PTP2C by p44i Iecreaus Its Phos- phatase Activity. To determine whether phosphorylation of PTP2C by p44nIaPk modified its enzymatic activity, 32P,- Added to labeled EGF receptor, previously shown to be dephospho- pellets: " rylated by PTP2C (10, 30), was used as a substrate. In brief, Origin extracts from untreated PC12 cells were incubated with antibody to PTP2C, or with nonimmune antibodies. Washed immunoprecipitates were exposed to buffer or to activated p44maPk as described above, except that unlabeled ATP was FIG. 2. (Left) Phosphorylation of immunopurified PTP2C by used. After washes, beads were then incubated with the p44PaPk. Cell extracts from serum-starved PC12 cells were incubated autophosphorylated EGF receptor and the resulting samples with antibody to PTP2C (lanes a, b, and d) or with nonimmune were subjected to SDS/PAGE followed by autoradiography antibodies (lane c). The immunoprecipitates were incubated for 45 (Fig. 3). Compared with the control incubation with nonim- min in the presence of kinase buffer alone (lane a) or with nonacti- mune antibodies (lane A), immunopurified PTP2C induced a vated (lane b) or activated (lanes c and d) p44maPk. Samples were 70%o decrease in the phosphorylation of the EGF receptors analyzed by SDS/PAGE. (Right) Phospho amino acid composition (lane B). In contrast, when PTP2C was phosphorylated by of PTP2C in lane d. Representative autoradiograms of four separate activated p44Pk prior to the phosphatase assay, the EGF experiments with similar results are shown. receptor was dephosphorylated by only 20% (compare lane D with lane C). Phospho amino acid analyses showed that, However, after 5 min of EGF treatment, a pronounced under our expenmental conditions, the EGF receptor was increase in threonine phosphorylation was observed along phosphorylated solely on tyrosine residues; consequently, with a small enrichment in serine phosphorylation. When the dephosphorylation took place on tyrosine only (Fig. 4). exposure to EGF was maintained for 45 min, the level of PTP2C Activity Is Inhibited Following EGF Treatment of PTP2C phosphorylation on both residues was comparable to PC12 Cells. We have shown so far that, in intact PC12 cells, that seen in untreated cells. In all cases, essentially no EGF leads to PTP2C phosphorylation on threonine residues phosphotyrosine was detected. and that, at least in the cell-free system, phosphorylation of PT2c Is a Substrate for p44hIP. The ability of p44maPk to PTP2C by p44mPk inhibits its enzymatic activity. We and phosphorylate PTP2C was tested in an in vitro kinase assay others previously reported that p44Iapk was activated tran-

kDa PC12 cells 206 -

EGF -_- * *@ @ FIG. 3. Dephosphorylation of receptor EGF receptors by PTP2C before and after phosphorylation by antibodies used for p44mapk. Extract from serum- immunopurification: control anti PTP2C control anti PTP2C 105 starved PC12 cells was incubated with nonimmune antibodies (lanes A and C) or antibody to PTP2C (lanes B and D). Immunoprecipi- - added to pellets: buffer activated p44mapk 70 tates were then treated for 45 min in the presence of activated p44maPk (lanes C and D) or in its absence (lanes A and B). PTP2C addition of 32P- EGF receptors activity was assayed with phos- 43 phorylated EGF receptors and an- alyzed by SDS/PAGE. A repre- sentative autoradiogram of three separate experiments with similar A B C D A B C D results is shown. Downloaded by guest on September 24, 2021 Biochemistry: Peraldi et al. Proc. NatL. Acad. Sci. USA 91 (1994) 5005

Pi - .W and 45 min with 0.1 j&M EGF and the cell extracts were subjected to immunoprecipitation with antibody to PTP2C. P-SER - Samples were incubated in the presence of 32P,-labeled EGF receptors and subjected to SDS/PAGE (Fig. 5). Compared P-THR - with control incubations with nonimmune antibodies, PTP2C from unstimulated cells induced a 65% dephosphorylation of the EGF receptor. This dephosphorylation was reduced to 15% in the presence ofPTP2C from cells exposed to EGF for 5 min. Finally, after 45 min of exposure to EGF, PTP2C P-TYR - I activity returned to the level (60%o dephosphorylation) seen in unstimulated cells. DISCUSSION In this paper, we have investigated the effects of EGF on PTP2C phosphorylation and activity in PC12 cells. In un- treated PC12 cells, PTP2C is phosphorylated mainly on serine residues. Exposure of these cells to EGF induced a transient increase in phosphothreonine residues and a small enrichment in phosphoserine. No tyrosine phosphorylation was detected, as was found by Kuhnd et al. (16) in 3T3-L1 Origin _- adipocytes. It remains to be determined whether the absence of phosphotyrosine was due to a lack of tyrosine phosphor- EGF receptor ylation per se or to a vigorous autodephosphorylation by the from conditions: A B C D phosphatase itself. Since the EGF-induced phosphorylation of PTP2C in PC12 cells followed the same time course as the FIG. 4. Phospho amino acid of in vitro analysis phosphorylated activation of p44tIvk by EGF (29, 30), we hypothesized that EGF receptors treated or untreated with PFP2C as described in Fig. 3 (lanes A-D). PTP2C could serve as a substrate for p44maPk. Using immu- nopurified PTP2C and recombinant p44maPk, we found this siently after EGF treatment ofPC12 cells (31, 32). Therefore, indeed to be the case in vitro. Further, phospho amino acid of PTP2C showed that the took it was important to investigate whether exposure of PC12 analyses phosphorylation cells to EGF could lead to modification in the activity of place on both threonine and serine residues. While it has been reported that the sequence Pro-Xaa-Ser/Thr-Pro is the op- PTP2C, and, if so, whether this effect had a similar transient timal motiffor MAP kinase phosphorylation (33), the minimal PC12 cells were treated for feature. For this purpose, 0, 5, consensus sequence appears to be Ser/Thr-Pro (26). Exam- ination of the amino acid sequence of PTP2C shows that the PC12 cells treated contains the three with EGF tor: ° 5 45 min phosphatase following putative phosphor- ylation sites for p44mPk: Ser-558 (Ser-Pro), and Thr-564 and kDa -566 (Pro-Cys-Thr-Pro-Thr-Pro). Considering our findings that PTP2C is phosphorylated by p44mal*, we think it is likely 206 that Ser-558 serves as one of the p44m1Pk phosphorylation sites. However, we do not know whether Thr-564 or Thr-566 or both, are phosphorylated by p44maPk. The serine/threo- EGF --a 0 :W..a -0 nine phosphorylation of PTP2C by p44m*k appears to be receptor physiologically relevant, since it is associated with a decrease in the activity of PTP2C both in vivo and in vitro. Inhibition by serine/threonine phosphorylation could represent a gen- 105 * eral mechanism ofregulation ofPTPs, as it has also been seen with PTP1B (34). Our data on the effect of EGF on PTP2C phosphorylation in PC12 cells could appear at variance with previous reports, 70 which found either no change in PTP2C activity (11, 14, 29) or a weak activation upon growth factor stimulation (13). However, these discrepancies might be explained as follows. (i) Feng et al. (14), who reported EGF- and PDGF-stimulated tyrosine , were studying Syp, an alterna- ipliv -i AD tively spliced mouse homologue of PTP2C which lacks the putative phosphorylation sites for p44mWk. (ii) In their study 43 - Added to EGF receptors: showing that EGF induced an increase in tyrosine phosphor- Pellets from ylation together with a small activation ofPTP2C, Vogel et al. C0 used cells the precipitates with serum: (13) overexpressing both EGF receptor and -o v) ;,>2~ PTP2C. While it is possible that this tyrosine phosphorylation could occur in nonoverexpressing cells, overexpression would favor the phosphorylation reaction. (iii) In our previ- FiG. 5. Activity of PIP2C obtained from PC12 cells treated with ous study (29), we did not find significant effects on the EGF. Serum-starved PC12 cells were treated forO, 5, and45 min with activity of purified recombinant PTP2C following phosphor- 0.1 pM EGF. Cell extracts were incubated with antibody to PTP2C MAP kinase. this be due to the or with nonimmune antibodies (control). The immunoprecipitates ylation by However, might were incubated with phosphorylated EGF receptors for 30 min, and low stoichiometry of phosphorylation obtained and differ- the reaction mixtures were subjected to SDS/PAGE. A representa- ences in substrates used. (iv) PC12, the cell line used in the tive autoradiogram ofthree separate experiments with similar results present study, displays a rather high level of p44mPk (35), is shown. which can be potently stimulated (>50-fold) by EGF (31). Downloaded by guest on September 24, 2021 5006 Biochemistry: Peraldi et al. Proc. Natl. Acad. Sci. USA 91 (1994) Hence, it is possible that the prevailing effect on PTP2C in 13. Vogel, W., Lammers, R., Huang, J. & Ullrich, A. (1993) PC12 cells is an inhibitory threonine/serine phosphorylation Science 259, 1611-1613. by 14. Feng, G.-S., Hui, C.-C. & Pawson, T. (1993) Science 259, mediated p44raPk. 1607-1610. The function of PTP2C is unknown, as the cellular sub- 15. Lechleider, R. J., Freeman, R. M. & Neel, B. G. (1993)J. Biol. strates have not been identified. We were unable to dephos- Chem. 268, 13434-13438. phorylate p44maPk by PTP2C (data not shown), suggesting 16. Kuhn6, M. R., Pawson, T., Lienhard, G. E. & Feng, G. S. that this phosphatase does not participate in a loop regulating (1993) J. Biol. Chem. 268, 11479-11481. the MAP kinase. If one assumes, based on this and other 17. Lechleider, R. J., Sugimoto, S., Bennet, A., Kashishian, A. S., Cooper, J. A., Shoelson, S. E., Walsh, C. T. & Neel, B. G. observations (10, 30), that the EGF receptor is indeed a target (1993) J. Biol. Chem. 268, 21478-21481. of PTP2C in intact cells, one could suggest that the EGF- 18. Gomez, N. & Cohen, P. (1991) Nature (London) 353, 170-173. induced inhibition ofPTP2C could serve as a means by which 19. Crews, C. M., Alessandrini, A. & Erikson, R. (1992) Science PC12 cells facilitate the EGF action. Admittedly, such a view 258, 478-480. might represent an oversimplification, as it is likely that 20. Seger, R., Ahn, N. G., Posada, J., Munar, E. S., Jensen, PTP2C could also act on a panel of other cellular targets. A. M., Cooper, J. A., Cobb, M. H. & Krebs, E. G. (1992) J. An outburst new Biol. Chem. 267, 14373-14381. of information has identified the MAP 21. Seger, R., Seger, D., Lozeman, F. J., Ahn, N. G., Graves, kinase signaling system as a major pathway linking the cell L. M., Campbell, J. S., Ericsson, A. M. & Krebs, E. G. (1992) membrane to the nucleus and regulating metabolism, growth, J. Biol. Chem. 267, 25628-25631. and differentiation (26, 36-38). A growing number of MAP 22. Haystead, T. A. J., Weiel, J. E., Litchfield, D. W., Tsukitani, kinase substrates have been identified in various subcellular Y., Fischer, E. H. & Krebs, E. G. (1990) J. Biol. Chem. 265, compartments, including the cell surface, the cytosol, and the 16571-16580. 23. Peraldi, P. & Van Obberghen, E. (1993) Eur. J. Biochem. 218, nucleus (26). These substrates include such diverse mole- 815-821. cules as various protein kinases acting upstream (39-41) or 24. Peraldi, P., Scimeca, J. C., Filloux, C. & Van Obberghen, E. downstream in the MAP kinase cascade (42, 43); the EGF (1993) Endocrinology 132, 2578-2585. receptor (44); tyrosine hydroxylase (45); Rab4, a small GTP- 25. Sun, H., Charles, C. H., Lau, L. F. & Tonks, N. K. (1993) Cell binding protein (46); and several transcription factors (33, 47, 75, 487-493. 48). Our present demonstration that MAP kinase regulates by 26. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556. phosphorylation the activity of PTP2C further emphasizes 27. Kyriakis, J. M., App, H., Zhang, X.-F., Banerjee, P. B., Brautigan, D. L., Rapp, U. R. & Avruch, J. (1992) Nature the universal role ofthe MAP kinase pathway in cell function. (London) 358, 417-421. We are grateful to Drs. M. Cormont, Y. Le Marchand Brustel, E. 28. Kamps, M. P. & Sefton, B. M. (1989) Anal. Biochem. 176, Van Obberghen-Schilling, J.-F. Tanti, and S. Giorgetti for discus- 22-27. sions and helpwith the manuscript. We thank Drs. Alfred Reszka and 29. Zhao, Z., Robert Larocque, Wan-Ting Ho, Fischer, E. H. & Jocelyn H. Wright for critical review of the manuscript. Dr. J. Shen, S.-H. (1994) J. Biol. Chem. 269, 8780-8785. Avruch (Diabetes Unit, Massachusetts General Hospital, Harvard 30. Sugimoto, S., Lechleider, R. J., Shoelson, S. E., Neel, B. G. Medical School, Boston) is acknowledged for providing bacteria & Walsh, C. T. (1993) J. Biol. Chem. 268, 2271-2276. expressing recombinant rat 44-kDa MAP kinase as a glutathione 31. Nguyen, T. T., Scimeca, J. C., Filloux, C., Peraldi, P., Car- S-transferase fusion protein. We thank Ms. J. Duch for secretarial pentier, J. L. & Van Obberghen, E. (1993) J. Biol. Chem. 268, assistance and Mr. G. Visciano for photographs. This work was 9803-9810. supported by Institut National de la Sant6 et de la Recherche 32. Gotoh, Y., Nishida, E., Yamashita, T., Hoshi, M., Kawakami, M6dicale; Universitd de Nice-Sophia-Antipolis; Association pour la M. & Sakai, H. (1990) Eur. J. Biochem. 193, 661-669. Recherche contre le Cancer, Grant 6760; Ligue Nationale Franqaise 33. Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, contre le Cancer, F6d6ration des Comit6s D6partementaux, Comite D. A., Seth, A., Abate, C., Curran, T. & Davis, R. J. (1991) J. D6partemental du Var, and Societe LiPHA (Lyon, France; Contract Biol. Chem. 266, 15277-15285. 93123); National Institutes ofHealth Grants DK07902 and GM42508; 34. Flint, A. J., Gebbink, M. F. G. B., Franza, B. R., Hill, D. E. and the Muscular Dystrophy Association of America. Z.Z. is a & Tonks, N. K. (1993) EMBO J. 12, 1937-1946. recipient of a Sugen research fellowship. 35. Boulton, T. G. & Cobb, M. H. (1991) Cell Regul. 2, 357-371. 36. Ahn, N. G., Seger, R. & Krebs, E. G. (1992) Curr. Opin. Cell 1. Ullrich, A. & Schlessinger, J. (1990) Cell 61, 203-212. Biol. 4, 9M-999. 2. Krebs, E. G. (1986) in The Enzymes, eds. Boyer, P. D. & 37. Crews, C. M. & Erikson, R. L. (1993) Cell 74, 215-217. Krebs, E. G. (Academic, Orlando, FL), Vol. 17, pp. 3-20. 38. Blenis, J. (1993) Proc. Natl. Acad. Sci. USA 90, 5889-5892. 3. Fischer, E. H., Charbonneau, H. & Tonks, N. K. (1991) Sci- 39. Anderson, N. G., Li, P., Marsden, L. A., Williams, N., Rob- ence 253, 401-406. erts, T. M. & Sturgill, T. W. (1991) Biochem. J. 277, 573-576. 4. Walton, K. M. & Dixon, J. E. (1993) Annu. Rev. Biochem. 62, 40. Lee, R.-M., Cobb, M. H. & Blackshear, P. J. (1992) J. Biol. 101-120. Chem. 267, 1088-1092. 5. Shen, S.-H., Bastien, L., Posner, B. I. & Chr6tien, P. (1991) 41. Matsuda, S., Gotoh, Y. & Nishida, E. (1993) J. Biol. Chem. Nature (London) 352, 736-739. 268, 3277-3281. 6. Plutzky, J., Neel, B. G. & Rosenberg, R. D. (1992) Proc. Natl. 42. Sturgill, T. W., Ray, L. B., Erikson, E. & Maller, J. L. (1988) Acad. Sci. USA 89, 1123-1127. Nature (London) 334, 715-718. 7. Yi, T., Cleveland, J. L. & Ihle, J. N. (1992) Mol. Cell. Biol. 12, 43. Ahn, N. G. & Krebs, E. G. (1990) J. Biol. Chem. 265, 11495- 836-846. 11501. 8. Matthew, R. J., Bowne, D. B., Flores, E. & Thomas, L. T. 44. Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, (1992) Mol. Cell. Biol. 12, 2396-2405. D. L. & Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276. 9. Plutzky, J., Neel, B. G., Rosenberg, R. D., Eddy, R. L., 45. Haycock, J. W., Ahn, N. G., Cobb, M. H. & Krebs, E. G. Byers, M. G., Jani-Sait, S. & Shows, T. B. (1992) Genomics 13, (1992) Proc. Natl. Acad. Sci. USA 89, 2365-2369. 869-872. 46. Cormont, M., Tanti, J.-F., Zahraoui, A., Van Obberghen, E. & 10. Ahmad, S., Banville, D., Zhao, Z., Fischer, E. H. & Shen, Le Marchand-Brustel, Y. (1994) Eur. J. Biochem. 219, 1081- S.-H. (1993) Proc. Natl. Acad. Sci. USA 90, 2197-2201. 1085. 11. Freeman, R. M., Plutzky, J. & Neel, B. G. (1992) Proc. Natl. 47. Gille, H., Sharrocks, A. D. & Shaw, P. E. (1992) Nature Acad. Sci. USA 89, 11239-11243. (London) 358, 414-417. 12. Adachi, M., Sekiya, M., Miyashi, T., Matsuno, K., Hinoda, Y., 48. Pulverer, B. J., Kyriakis, J. M., Avruch, J., Nikolakaki, E. & Imai, K. & Yachi, A. (1992) FERS Lett. 314, 335-339. Woodgett, J. R. (1991) Nature (London) 353, 670-674. Downloaded by guest on September 24, 2021