Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5002-5006, May 1994 Biochemistry Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase 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 phosphorylation 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 serine 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 threonine 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. Amino acid 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 kinases 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, proteins 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.