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Identification of Epidermal Growth Factor Receptor as a Target of Cdc25A Protein Phosphatase

2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês

10.1074/jbc.m201097200

ISSN

1083-351X

Autores

Ziqiu Wang, Meifang Wang, John S. Lazo, Brian I. Carr,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

Cdc25A, a dual-specificity protein phosphatase, plays a critical role in cell cycle progression. Although cyclin-dependent kinases are established substrates, Cdc25A may also affect other proteins. We have shown here that Cdc25A interacts with epidermal growth factor receptor (EGFR) both physically and functionally in Hep3B human hepatoma cells. Cdc25A inhibitor Cpd 5, a vitamin K analog, inhibited Cdc25A activity in the Cdc25A-EGFR immunocomplex and consequently caused prolonged EGFR tyrosine phosphorylation. Both purified GST-Cdc25A protein and endogenous Hep3B cellular Cdc25A dephosphorylated tyrosine-phosphorylated EGFR, and Cpd 5 antagonized the phosphatase activity of Cdc25A. A functional Cdc25A-EGFR interaction was seen in NR-6 fibroblasts expressing ectopic EGFR but not with a receptor lacking the C terminus or a mutated kinase domain. These data link the cell cycle control Cdc25A phosphatase to an EGFR-linked mitogenic signaling pathway specifically involving EGFR dephosphorylation. Cdc25A, a dual-specificity protein phosphatase, plays a critical role in cell cycle progression. Although cyclin-dependent kinases are established substrates, Cdc25A may also affect other proteins. We have shown here that Cdc25A interacts with epidermal growth factor receptor (EGFR) both physically and functionally in Hep3B human hepatoma cells. Cdc25A inhibitor Cpd 5, a vitamin K analog, inhibited Cdc25A activity in the Cdc25A-EGFR immunocomplex and consequently caused prolonged EGFR tyrosine phosphorylation. Both purified GST-Cdc25A protein and endogenous Hep3B cellular Cdc25A dephosphorylated tyrosine-phosphorylated EGFR, and Cpd 5 antagonized the phosphatase activity of Cdc25A. A functional Cdc25A-EGFR interaction was seen in NR-6 fibroblasts expressing ectopic EGFR but not with a receptor lacking the C terminus or a mutated kinase domain. These data link the cell cycle control Cdc25A phosphatase to an EGFR-linked mitogenic signaling pathway specifically involving EGFR dephosphorylation. Phosphorylation and dephosphorylation of proteins provide a well studied regulatory mechanism for eliciting major changes in cell growth and differentiation. Among the proteins involved are cell surface receptors that are endowed with intrinsic protein-tyrosine kinase activity. These receptor-tyrosine kinases (RTKs) 1The abbreviations used are: RTK(s)receptor-tyrosine kinase(s)PTPase(s)protein-tyrosine phosphatase(s)EGFepidermal growth factorEGFREGF receptorMAPKmitogen-activated protein kinaseGSTglutathione S- transferaseERKextracellular signal-related kinase1The abbreviations used are: RTK(s)receptor-tyrosine kinase(s)PTPase(s)protein-tyrosine phosphatase(s)EGFepidermal growth factorEGFREGF receptorMAPKmitogen-activated protein kinaseGSTglutathione S- transferaseERKextracellular signal-related kinase play an important role in the control of many fundamental cellular processes including cell cycle control, survival, metabolism, and differentiation (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). Tyrosine-phosphorylated growth factor receptors are rapidly inactivated by dephosphorylation, a process that is believed to negatively modulate signaling activity (2Lammers R. Bossenmaier B. Cool DE. Tonks N.K. Schlessinger J. Fischer E.H. Ullrich A. J. Biol. Chem. 1993; 268: 22456-22462Abstract Full Text PDF PubMed Google Scholar, 3Kulas D.T. Goldstein B.J. Mooney R.A. J. Biol. Chem. 1996; 271: 748-754Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The identity of many of the protein-tyrosine phosphatases (PTPases) involved in receptor dephosphorylation is unknown. PTPases constitute a diverse family of enzymes that can be divided into several subgroups, including receptor PTPases and non-receptor PTPases (4Fischer E.H. Charbonneau H. Tonks N.K. Science. 1991; 253: 401-406Crossref PubMed Scopus (863) Google Scholar, 5Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). All PTPases contain an essential cysteine residue in the enzyme active site, and this essential cysteine is the target for specific modification by various sulfhydryl-alkylating reagents (6Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (157) Google Scholar, 7Zhou G. Denu J.M., Wu, L. Dixon J.E. J. Biol. Chem. 1994; 269: 28084-28090Abstract Full Text PDF PubMed Google Scholar). Recently, a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5), has been found to be a potent growth inhibitor for both normal rat hepatocytes and hepatoma cells. This growth inhibition is associated with enhanced and prolonged EGFR tyrosine phosphorylation (8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar, 9Nishikawa Y. Wang Z. Kerns J. Wilcox C.S. Carr B.I. J. Biol. Chem. 1999; 274: 34803-34810Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 10Ni R. Nishikawa Y. Carr B.I. J. Biol. Chem. 1998; 273: 9906-9911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cpd 5 is able to arylate cellular thiols or thiol-dependent proteins. Thus, PTPases are a likely group of target proteins for Cpd 5. The inactivation of PTPases by Cpd 5 may cause an imbalance of EGFR tyrosine phosphorylation and dephosphorylation and thus perturb the regulation of cell growth and other cellular functions.Cdc25 phosphatases are a PTPase subfamily; they contain a catalytic cysteine residue and are essential regulators of cell cycle transitions. In mammalian cells, three Cdc25-related proteins have been identified (11Sadhu K. Reed S.I. Richardson H. Russell P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5139-5143Crossref PubMed Scopus (283) Google Scholar, 12Galaktionov K. Beach D. Cell. 1991; 67: 1181-1194Abstract Full Text PDF PubMed Scopus (418) Google Scholar). Among them, Cdc25B and Cdc25C appear to regulate progression from G2 to M phase while Cdc25A is required for S phase entry, and its overexpression leads to an acceleration in S phase entry (13Millar J.B.A. Blevitt J. Gerace L. Sadhu K. Featherstone C. Russell P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10500-10504Crossref PubMed Scopus (194) Google Scholar, 14Karlsson C. Katich S. Hagting A. Hoffmann I. Pines J. J. Cell Biol. 1999; 146: 573-583Crossref PubMed Scopus (144) Google Scholar, 15Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (253) Google Scholar, 16Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (422) Google Scholar). Recent evidence suggests that Cdc25A may also have a role in the initiation of mitosis (17Molinari M. Mercurio C. Dominguez J. Goubin F. Draetta G.F. EMBO Rep. 2000; 1: 71-79Crossref PubMed Scopus (171) Google Scholar). Although Cdc25A clearly can dephosphorylate the cyclin-dependent kinases including Cdk2/cyclin A(E) and Cdk4 (Cdk6)/cyclin D, the identities of other potential substrates remain unclear (18Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Crossref PubMed Scopus (552) Google Scholar, 19Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar, 20Draetta G. Eckstein J. Biochim. Biophys. Acta. 1997; 1332: M53-M63PubMed Google Scholar, 21Millar J.B.A. McGowan C.H. Lenaers G. Jones R. Russell P. EMBO J. 1991; 10: 4301-4309Crossref PubMed Scopus (250) Google Scholar, 22Jinno S. Suto A. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar, 23Hoffmann I. Clarke P. Marcote M. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-56Crossref PubMed Scopus (562) Google Scholar, 24Galaktionov K. Chen X. Beach D. Nature. 1996; 382: 511-517Crossref PubMed Scopus (643) Google Scholar). Recent work indicates that Cdc25A can act on substrates other than Cdks because it dephosphorylates the homeodomain transcription factor cut, leading to a decrease in p21 promoter activity (25Coqueret O. Berube G. Nepveu A. EMBO J. 1998; 17: 4680-4694Crossref PubMed Scopus (133) Google Scholar). Furthermore, Cdc25A was found to interact with and dephosphorylate the proto-oncogene Raf-1 on tyrosine residues, resulting in a significant decrease in Raf-1 kinase activity (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). That Cdc25A is not highly promiscuous was suggested by the work of Zou et al. (28Zou X. Tsutsui T. Ray D. Blomquist J.F. Ichijo H. Ucker D.S. Kiyokawa H. Mol. Cell. Biol. 2001; 21: 4818-4828Crossref PubMed Scopus (87) Google Scholar), which used a yeast two-hybrid system to screen >106 clones and found Cdc25A bound only to 14-3-3ζ, apoptosis signal-regulating kinase 1, and three other as yet unidentified proteins.We have previously shown that Cpd 5 causes persistent EGFR tyrosine phosphorylation, which is related to inhibition of EGFR tyrosine phosphatases. However, the target PTPases are still not identified. We examined SH-PTP1 and SH-PTP2, two prototype PTPases for EGFR dephosphorylation, and found that their activity was not inhibited by Cpd 5 (Ref. 8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar). 2Z. Wang and B. I. Carr, unpublished data.2Z. Wang and B. I. Carr, unpublished data. There is growing evidence that the Cdc25A dual-specificity phosphatase plays an important role in regulating signal transduction pathways and cell growth, and some recent reports show that vitamin K3 and Cpd 5 inhibit cellular Cdc25 activity (29Wu FY-H. Sun T-P. Eur. J. Cancer. 1999; 35: 1388-1393Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 30Rice R. Rusnak J.M. Yokokawa F. Yokokawa S. Messner D.J. Boynton A.L. Wipf P. Lazo J.S. Biochemistry. 1997; 36: 15965-15974Crossref PubMed Scopus (86) Google Scholar, 31Tamura K. Southwick E.C. Kerns J. Rosi K.S. Carr B.I. Wilcox C.S. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar). Therefore, we considered that Cpd 5 would be a useful tool to study EGFR and Cdc25A interactions. Here we provide evidence that Cdc25A physically and functionally interacted with EGFR and dephosphorylated EGFR that had been tyrosine-phosphorylated in vitro and in culture. Furthermore, in EGFR kinase-mutated and its C-terminal-deleted NR-6 fibroblast cells, no Cdc25A-EGFR functional interaction was found. The inhibition of Cdc25A activity by Cpd 5 caused EGFR hyperphosphorylation. These data identify EGFR as a substrate for Cdc25A phosphatase.DISCUSSIONThe activated and autophosphorylated growth factor receptors are subject to a rapid dephosphorylation by PTPases (36Swarup G. Cohen S. Garbers D.L. Biochem. Biophys. Res. Commun. 1982; 107: 1104-1109Crossref PubMed Scopus (568) Google Scholar, 37Bohmer F.D. Bohmer S.A. Heldin C.H. FEBS Lett. 1993; 331: 276-280Crossref PubMed Scopus (14) Google Scholar). Receptor dephosphorylation is believed to represent a major mechanism of negative regulation of receptor function, and the identification of the involved PTPases is therefore important for understanding receptor signaling. We have previously shown that a PTPase inhibitor, Cpd 5, can strongly activate EGFR tyrosine phosphorylation, which is perhaps paradoxically related to cell growth inhibition (8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar). We excluded two prototype EGFR phosphatases, SH-PTP1 and SH-PTP2, as candidate targets for Cpd 5, because SH-PTP1 was not expressed in Hep3B cells and SH-PTP2 did not dephosphorylate EGFR (data not shown). Because our previous studies have shown that Cdc25A activity was inhibited by Cpd 5 in vitro and in cell cultures (31Tamura K. Southwick E.C. Kerns J. Rosi K.S. Carr B.I. Wilcox C.S. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar, 35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar), we examined the relationship between EGFR and Cdc25A. Cdc25A belongs to the Cdc25 phosphatase family in human cells and is well known as a G1/S and G2/M phase cell cycle regulator that catalyzes dephosphorylation and activation of cyclin-Cdk through removal of the inhibitory phosphates (17Molinari M. Mercurio C. Dominguez J. Goubin F. Draetta G.F. EMBO Rep. 2000; 1: 71-79Crossref PubMed Scopus (171) Google Scholar, 22Jinno S. Suto A. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar). However, the nature of its substrates remains unclear. Besides the likely substrates of cyclin-Cdk, it has been reported recently that Cdc25A binds to and dephosphorylates the homeodomain transcription factor cut (25Coqueret O. Berube G. Nepveu A. EMBO J. 1998; 17: 4680-4694Crossref PubMed Scopus (133) Google Scholar) and phosphoprotein Raf-1 (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). We have reported here for the first time that EGFR is likely to be a substrate of Cdc25A. This conclusion is based on the following findings. First, Cdc25A co-immunoprecipitated with EGFR in Cpd 5-treated Hep3B cells, and GST-Cdc25A directly bound to purified EGFR protein in vitro. Second, Cpd 5, a known inhibitor of Cdc25A phosphatase activity, induced a prolonged EGFR tyrosine phosphorylation. By contrast, in Hep3B cells expressing an inactivated Cdc25A, no EGFR tyrosine phosphorylation was found after the treatment with Cpd 5. This result is consistent with the report that the inactive mutant Cdc25A-C430S protein strongly interacts with both cyclin A-Cdk2 and cyclin E-Cdk2 but does not lead to an activation of Cdk2 kinase activity (38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Third, both purified GST-Cdc25A and endogenous Hep3B cellular Cdc25A dephosphorylated EGFR tyrosine phosphorylation in vitro and in cell culture; their dephosphorylation activity can be inhibited by Cpd 5, demonstrating that EGFR is a direct target of Cdc25A. Finally, in EGFR-transfected fibroblast NR-6 cells, Cdc25A was found to bind to wild-type and kinase-mutated EGFR but not to C-terminal-deleted EGFR. Furthermore, only wild-type EGFR was able to be tyrosine-phosphorylated with Cpd 5 treatment. These results suggest that Cdc25A binds to the C-terminal of EGFR and that EGFR kinase activity may be important to the action of Cdc25A on EGFR dephosphorylationIt seems that EGFR dephosphorylation by Cdc25A is selective, because Cdc25A has no effect on insulin receptor tyrosine dephosphorylation. This selectivity is probably because of the structural differences between EGFR and insulin receptor. Most RTKs, such as EGFR and platelet-derived growth factor receptor, are monomers in the cell membrane. Ligand binding induces dimerization of these receptors (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). However, members of the insulin receptor family are disulfide-linked dimers of two polypeptide chains forming an α2β2 heterodimer (39Van Obberghen E. Diabetologia. 1994; 27: S125-S134Crossref Scopus (60) Google Scholar). Insulin binding to the extracellular domain of the insulin receptor induces a rearrangement in the quaternary heterotetrameric structure that leads to increased autophosphorylation of the cytoplasmic domain (40Hubbard S.R. Mohhammadi M. Schlessinger J. J. Biol. Chem. 1998; 273: 11987-11990Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). This structure may not be suitable for Cdc25A binding. The fact that the Cdc25A inhibitor Cpd 5 cannot induce insulin receptor tyrosine phosphorylation further supports our hypothesis. In the Cdc25 family, only Cdc25A showed marked tyrosine phosphatase activity directed against EGFR. Our results are consistent with those previous findings that Cdc25A is a more potent tyrosine phosphatase than Cdc25B or Cdc25C when Raf-1 and Cdk are used as the substrates (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar, 38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The mechanisms for this discrepancy have not yet been elucidated. One possibility is that the Cdc25 phosphatase activity is regulated by extensive phosphorylation of the N-terminal regulatory domain, since the alignment of all known dual-specific protein-tyrosine phosphatases shows that they are very different from each other in terms of length and amino acid sequence outside the putative catalytic domain (38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,41Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (335) Google Scholar).The Cdc25 protein phosphatase family has long been regarded as only a cell cycle regulator. However, recent studies have shown that Cdc25A may also play a central role in regulating mitogenic signal transduction pathways. For instance, it has been reported that Cdc25A can physically associate with Raf-1 and regulates Raf-1 tyrosine phosphorylation and its activity (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). The fact that Raf-1 kinase can form complexes with the cell cycle activator Cdc25A provides strong evidence that signal transduction pathways are linked with the cell cycle directly. In HeLa cells, overexpression of Cdc25A in whole cells induced ERK dephosphorylation, and the Cdc25A inhibitor Cpd 5 restored ERK phosphorylation and nuclear translocation, demonstrating that Cdc25A regulated endogenous ERK phosphorylation status in whole cells (34Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We also found that Cpd 5-inhibited Cdc25A activity could contribute to Cdk4 tyrosine phosphorylation and subsequent cell cycle block and cell growth inhibition (35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar). In this study, we again used Cpd 5 as a tool to reveal that Cdc25A was an EGFR phosphatase and that inhibition of Cdc25A activity by Cpd 5 caused prolonged EGFR tyrosine phosphorylation. These data suggest that Cdc25A acts as the phosphatase for several different substrates in the MAPK pathway and that Cpd 5-induced activation of the MAPK pathway can be triggered by either upstream activation of EGFR or Raf-1 or by direct inhibition of ERK phosphatase Cdc25A. Taken together, our data establish Cdc25A protein phosphatase as a key molecule in regulating and linking cell cycle progression and signal transduction pathway. Inhibition of Cdc25A activity by Cpd 5 not only overstimulates the MAPK pathway from membrane receptor to nuclear, which down-regulates gene transcription, but also directly inhibits cell cycle progression, eventually leading to growth inhibition and cell death. Thus, finding an efficient Cdc25A inhibitor may be of significant importance in controlling cancer cell growth and proliferation. Phosphorylation and dephosphorylation of proteins provide a well studied regulatory mechanism for eliciting major changes in cell growth and differentiation. Among the proteins involved are cell surface receptors that are endowed with intrinsic protein-tyrosine kinase activity. These receptor-tyrosine kinases (RTKs) 1The abbreviations used are: RTK(s)receptor-tyrosine kinase(s)PTPase(s)protein-tyrosine phosphatase(s)EGFepidermal growth factorEGFREGF receptorMAPKmitogen-activated protein kinaseGSTglutathione S- transferaseERKextracellular signal-related kinase1The abbreviations used are: RTK(s)receptor-tyrosine kinase(s)PTPase(s)protein-tyrosine phosphatase(s)EGFepidermal growth factorEGFREGF receptorMAPKmitogen-activated protein kinaseGSTglutathione S- transferaseERKextracellular signal-related kinase play an important role in the control of many fundamental cellular processes including cell cycle control, survival, metabolism, and differentiation (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). Tyrosine-phosphorylated growth factor receptors are rapidly inactivated by dephosphorylation, a process that is believed to negatively modulate signaling activity (2Lammers R. Bossenmaier B. Cool DE. Tonks N.K. Schlessinger J. Fischer E.H. Ullrich A. J. Biol. Chem. 1993; 268: 22456-22462Abstract Full Text PDF PubMed Google Scholar, 3Kulas D.T. Goldstein B.J. Mooney R.A. J. Biol. Chem. 1996; 271: 748-754Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The identity of many of the protein-tyrosine phosphatases (PTPases) involved in receptor dephosphorylation is unknown. PTPases constitute a diverse family of enzymes that can be divided into several subgroups, including receptor PTPases and non-receptor PTPases (4Fischer E.H. Charbonneau H. Tonks N.K. Science. 1991; 253: 401-406Crossref PubMed Scopus (863) Google Scholar, 5Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). All PTPases contain an essential cysteine residue in the enzyme active site, and this essential cysteine is the target for specific modification by various sulfhydryl-alkylating reagents (6Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (157) Google Scholar, 7Zhou G. Denu J.M., Wu, L. Dixon J.E. J. Biol. Chem. 1994; 269: 28084-28090Abstract Full Text PDF PubMed Google Scholar). Recently, a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5), has been found to be a potent growth inhibitor for both normal rat hepatocytes and hepatoma cells. This growth inhibition is associated with enhanced and prolonged EGFR tyrosine phosphorylation (8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar, 9Nishikawa Y. Wang Z. Kerns J. Wilcox C.S. Carr B.I. J. Biol. Chem. 1999; 274: 34803-34810Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 10Ni R. Nishikawa Y. Carr B.I. J. Biol. Chem. 1998; 273: 9906-9911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cpd 5 is able to arylate cellular thiols or thiol-dependent proteins. Thus, PTPases are a likely group of target proteins for Cpd 5. The inactivation of PTPases by Cpd 5 may cause an imbalance of EGFR tyrosine phosphorylation and dephosphorylation and thus perturb the regulation of cell growth and other cellular functions. receptor-tyrosine kinase(s) protein-tyrosine phosphatase(s) epidermal growth factor EGF receptor mitogen-activated protein kinase glutathione S- transferase extracellular signal-related kinase receptor-tyrosine kinase(s) protein-tyrosine phosphatase(s) epidermal growth factor EGF receptor mitogen-activated protein kinase glutathione S- transferase extracellular signal-related kinase Cdc25 phosphatases are a PTPase subfamily; they contain a catalytic cysteine residue and are essential regulators of cell cycle transitions. In mammalian cells, three Cdc25-related proteins have been identified (11Sadhu K. Reed S.I. Richardson H. Russell P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5139-5143Crossref PubMed Scopus (283) Google Scholar, 12Galaktionov K. Beach D. Cell. 1991; 67: 1181-1194Abstract Full Text PDF PubMed Scopus (418) Google Scholar). Among them, Cdc25B and Cdc25C appear to regulate progression from G2 to M phase while Cdc25A is required for S phase entry, and its overexpression leads to an acceleration in S phase entry (13Millar J.B.A. Blevitt J. Gerace L. Sadhu K. Featherstone C. Russell P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10500-10504Crossref PubMed Scopus (194) Google Scholar, 14Karlsson C. Katich S. Hagting A. Hoffmann I. Pines J. J. Cell Biol. 1999; 146: 573-583Crossref PubMed Scopus (144) Google Scholar, 15Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (253) Google Scholar, 16Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (422) Google Scholar). Recent evidence suggests that Cdc25A may also have a role in the initiation of mitosis (17Molinari M. Mercurio C. Dominguez J. Goubin F. Draetta G.F. EMBO Rep. 2000; 1: 71-79Crossref PubMed Scopus (171) Google Scholar). Although Cdc25A clearly can dephosphorylate the cyclin-dependent kinases including Cdk2/cyclin A(E) and Cdk4 (Cdk6)/cyclin D, the identities of other potential substrates remain unclear (18Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Crossref PubMed Scopus (552) Google Scholar, 19Iavarone A. Massague J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (328) Google Scholar, 20Draetta G. Eckstein J. Biochim. Biophys. Acta. 1997; 1332: M53-M63PubMed Google Scholar, 21Millar J.B.A. McGowan C.H. Lenaers G. Jones R. Russell P. EMBO J. 1991; 10: 4301-4309Crossref PubMed Scopus (250) Google Scholar, 22Jinno S. Suto A. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar, 23Hoffmann I. Clarke P. Marcote M. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-56Crossref PubMed Scopus (562) Google Scholar, 24Galaktionov K. Chen X. Beach D. Nature. 1996; 382: 511-517Crossref PubMed Scopus (643) Google Scholar). Recent work indicates that Cdc25A can act on substrates other than Cdks because it dephosphorylates the homeodomain transcription factor cut, leading to a decrease in p21 promoter activity (25Coqueret O. Berube G. Nepveu A. EMBO J. 1998; 17: 4680-4694Crossref PubMed Scopus (133) Google Scholar). Furthermore, Cdc25A was found to interact with and dephosphorylate the proto-oncogene Raf-1 on tyrosine residues, resulting in a significant decrease in Raf-1 kinase activity (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). That Cdc25A is not highly promiscuous was suggested by the work of Zou et al. (28Zou X. Tsutsui T. Ray D. Blomquist J.F. Ichijo H. Ucker D.S. Kiyokawa H. Mol. Cell. Biol. 2001; 21: 4818-4828Crossref PubMed Scopus (87) Google Scholar), which used a yeast two-hybrid system to screen >106 clones and found Cdc25A bound only to 14-3-3ζ, apoptosis signal-regulating kinase 1, and three other as yet unidentified proteins. We have previously shown that Cpd 5 causes persistent EGFR tyrosine phosphorylation, which is related to inhibition of EGFR tyrosine phosphatases. However, the target PTPases are still not identified. We examined SH-PTP1 and SH-PTP2, two prototype PTPases for EGFR dephosphorylation, and found that their activity was not inhibited by Cpd 5 (Ref. 8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar). 2Z. Wang and B. I. Carr, unpublished data.2Z. Wang and B. I. Carr, unpublished data. There is growing evidence that the Cdc25A dual-specificity phosphatase plays an important role in regulating signal transduction pathways and cell growth, and some recent reports show that vitamin K3 and Cpd 5 inhibit cellular Cdc25 activity (29Wu FY-H. Sun T-P. Eur. J. Cancer. 1999; 35: 1388-1393Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 30Rice R. Rusnak J.M. Yokokawa F. Yokokawa S. Messner D.J. Boynton A.L. Wipf P. Lazo J.S. Biochemistry. 1997; 36: 15965-15974Crossref PubMed Scopus (86) Google Scholar, 31Tamura K. Southwick E.C. Kerns J. Rosi K.S. Carr B.I. Wilcox C.S. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar). Therefore, we considered that Cpd 5 would be a useful tool to study EGFR and Cdc25A interactions. Here we provide evidence that Cdc25A physically and functionally interacted with EGFR and dephosphorylated EGFR that had been tyrosine-phosphorylated in vitro and in culture. Furthermore, in EGFR kinase-mutated and its C-terminal-deleted NR-6 fibroblast cells, no Cdc25A-EGFR functional interaction was found. The inhibition of Cdc25A activity by Cpd 5 caused EGFR hyperphosphorylation. These data identify EGFR as a substrate for Cdc25A phosphatase. DISCUSSIONThe activated and autophosphorylated growth factor receptors are subject to a rapid dephosphorylation by PTPases (36Swarup G. Cohen S. Garbers D.L. Biochem. Biophys. Res. Commun. 1982; 107: 1104-1109Crossref PubMed Scopus (568) Google Scholar, 37Bohmer F.D. Bohmer S.A. Heldin C.H. FEBS Lett. 1993; 331: 276-280Crossref PubMed Scopus (14) Google Scholar). Receptor dephosphorylation is believed to represent a major mechanism of negative regulation of receptor function, and the identification of the involved PTPases is therefore important for understanding receptor signaling. We have previously shown that a PTPase inhibitor, Cpd 5, can strongly activate EGFR tyrosine phosphorylation, which is perhaps paradoxically related to cell growth inhibition (8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar). We excluded two prototype EGFR phosphatases, SH-PTP1 and SH-PTP2, as candidate targets for Cpd 5, because SH-PTP1 was not expressed in Hep3B cells and SH-PTP2 did not dephosphorylate EGFR (data not shown). Because our previous studies have shown that Cdc25A activity was inhibited by Cpd 5 in vitro and in cell cultures (31Tamura K. Southwick E.C. Kerns J. Rosi K.S. Carr B.I. Wilcox C.S. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar, 35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar), we examined the relationship between EGFR and Cdc25A. Cdc25A belongs to the Cdc25 phosphatase family in human cells and is well known as a G1/S and G2/M phase cell cycle regulator that catalyzes dephosphorylation and activation of cyclin-Cdk through removal of the inhibitory phosphates (17Molinari M. Mercurio C. Dominguez J. Goubin F. Draetta G.F. EMBO Rep. 2000; 1: 71-79Crossref PubMed Scopus (171) Google Scholar, 22Jinno S. Suto A. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar). However, the nature of its substrates remains unclear. Besides the likely substrates of cyclin-Cdk, it has been reported recently that Cdc25A binds to and dephosphorylates the homeodomain transcription factor cut (25Coqueret O. Berube G. Nepveu A. EMBO J. 1998; 17: 4680-4694Crossref PubMed Scopus (133) Google Scholar) and phosphoprotein Raf-1 (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). We have reported here for the first time that EGFR is likely to be a substrate of Cdc25A. This conclusion is based on the following findings. First, Cdc25A co-immunoprecipitated with EGFR in Cpd 5-treated Hep3B cells, and GST-Cdc25A directly bound to purified EGFR protein in vitro. Second, Cpd 5, a known inhibitor of Cdc25A phosphatase activity, induced a prolonged EGFR tyrosine phosphorylation. By contrast, in Hep3B cells expressing an inactivated Cdc25A, no EGFR tyrosine phosphorylation was found after the treatment with Cpd 5. This result is consistent with the report that the inactive mutant Cdc25A-C430S protein strongly interacts with both cyclin A-Cdk2 and cyclin E-Cdk2 but does not lead to an activation of Cdk2 kinase activity (38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Third, both purified GST-Cdc25A and endogenous Hep3B cellular Cdc25A dephosphorylated EGFR tyrosine phosphorylation in vitro and in cell culture; their dephosphorylation activity can be inhibited by Cpd 5, demonstrating that EGFR is a direct target of Cdc25A. Finally, in EGFR-transfected fibroblast NR-6 cells, Cdc25A was found to bind to wild-type and kinase-mutated EGFR but not to C-terminal-deleted EGFR. Furthermore, only wild-type EGFR was able to be tyrosine-phosphorylated with Cpd 5 treatment. These results suggest that Cdc25A binds to the C-terminal of EGFR and that EGFR kinase activity may be important to the action of Cdc25A on EGFR dephosphorylationIt seems that EGFR dephosphorylation by Cdc25A is selective, because Cdc25A has no effect on insulin receptor tyrosine dephosphorylation. This selectivity is probably because of the structural differences between EGFR and insulin receptor. Most RTKs, such as EGFR and platelet-derived growth factor receptor, are monomers in the cell membrane. Ligand binding induces dimerization of these receptors (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). However, members of the insulin receptor family are disulfide-linked dimers of two polypeptide chains forming an α2β2 heterodimer (39Van Obberghen E. Diabetologia. 1994; 27: S125-S134Crossref Scopus (60) Google Scholar). Insulin binding to the extracellular domain of the insulin receptor induces a rearrangement in the quaternary heterotetrameric structure that leads to increased autophosphorylation of the cytoplasmic domain (40Hubbard S.R. Mohhammadi M. Schlessinger J. J. Biol. Chem. 1998; 273: 11987-11990Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). This structure may not be suitable for Cdc25A binding. The fact that the Cdc25A inhibitor Cpd 5 cannot induce insulin receptor tyrosine phosphorylation further supports our hypothesis. In the Cdc25 family, only Cdc25A showed marked tyrosine phosphatase activity directed against EGFR. Our results are consistent with those previous findings that Cdc25A is a more potent tyrosine phosphatase than Cdc25B or Cdc25C when Raf-1 and Cdk are used as the substrates (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar, 38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The mechanisms for this discrepancy have not yet been elucidated. One possibility is that the Cdc25 phosphatase activity is regulated by extensive phosphorylation of the N-terminal regulatory domain, since the alignment of all known dual-specific protein-tyrosine phosphatases shows that they are very different from each other in terms of length and amino acid sequence outside the putative catalytic domain (38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,41Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (335) Google Scholar).The Cdc25 protein phosphatase family has long been regarded as only a cell cycle regulator. However, recent studies have shown that Cdc25A may also play a central role in regulating mitogenic signal transduction pathways. For instance, it has been reported that Cdc25A can physically associate with Raf-1 and regulates Raf-1 tyrosine phosphorylation and its activity (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). The fact that Raf-1 kinase can form complexes with the cell cycle activator Cdc25A provides strong evidence that signal transduction pathways are linked with the cell cycle directly. In HeLa cells, overexpression of Cdc25A in whole cells induced ERK dephosphorylation, and the Cdc25A inhibitor Cpd 5 restored ERK phosphorylation and nuclear translocation, demonstrating that Cdc25A regulated endogenous ERK phosphorylation status in whole cells (34Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We also found that Cpd 5-inhibited Cdc25A activity could contribute to Cdk4 tyrosine phosphorylation and subsequent cell cycle block and cell growth inhibition (35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar). In this study, we again used Cpd 5 as a tool to reveal that Cdc25A was an EGFR phosphatase and that inhibition of Cdc25A activity by Cpd 5 caused prolonged EGFR tyrosine phosphorylation. These data suggest that Cdc25A acts as the phosphatase for several different substrates in the MAPK pathway and that Cpd 5-induced activation of the MAPK pathway can be triggered by either upstream activation of EGFR or Raf-1 or by direct inhibition of ERK phosphatase Cdc25A. Taken together, our data establish Cdc25A protein phosphatase as a key molecule in regulating and linking cell cycle progression and signal transduction pathway. Inhibition of Cdc25A activity by Cpd 5 not only overstimulates the MAPK pathway from membrane receptor to nuclear, which down-regulates gene transcription, but also directly inhibits cell cycle progression, eventually leading to growth inhibition and cell death. Thus, finding an efficient Cdc25A inhibitor may be of significant importance in controlling cancer cell growth and proliferation. The activated and autophosphorylated growth factor receptors are subject to a rapid dephosphorylation by PTPases (36Swarup G. Cohen S. Garbers D.L. Biochem. Biophys. Res. Commun. 1982; 107: 1104-1109Crossref PubMed Scopus (568) Google Scholar, 37Bohmer F.D. Bohmer S.A. Heldin C.H. FEBS Lett. 1993; 331: 276-280Crossref PubMed Scopus (14) Google Scholar). Receptor dephosphorylation is believed to represent a major mechanism of negative regulation of receptor function, and the identification of the involved PTPases is therefore important for understanding receptor signaling. We have previously shown that a PTPase inhibitor, Cpd 5, can strongly activate EGFR tyrosine phosphorylation, which is perhaps paradoxically related to cell growth inhibition (8Wang Z. Wang M. Carr B.I. J. Cell. Physiol. 2000; 183: 338-346Crossref PubMed Scopus (29) Google Scholar). We excluded two prototype EGFR phosphatases, SH-PTP1 and SH-PTP2, as candidate targets for Cpd 5, because SH-PTP1 was not expressed in Hep3B cells and SH-PTP2 did not dephosphorylate EGFR (data not shown). Because our previous studies have shown that Cdc25A activity was inhibited by Cpd 5 in vitro and in cell cultures (31Tamura K. Southwick E.C. Kerns J. Rosi K.S. Carr B.I. Wilcox C.S. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar, 35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar), we examined the relationship between EGFR and Cdc25A. Cdc25A belongs to the Cdc25 phosphatase family in human cells and is well known as a G1/S and G2/M phase cell cycle regulator that catalyzes dephosphorylation and activation of cyclin-Cdk through removal of the inhibitory phosphates (17Molinari M. Mercurio C. Dominguez J. Goubin F. Draetta G.F. EMBO Rep. 2000; 1: 71-79Crossref PubMed Scopus (171) Google Scholar, 22Jinno S. Suto A. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar). However, the nature of its substrates remains unclear. Besides the likely substrates of cyclin-Cdk, it has been reported recently that Cdc25A binds to and dephosphorylates the homeodomain transcription factor cut (25Coqueret O. Berube G. Nepveu A. EMBO J. 1998; 17: 4680-4694Crossref PubMed Scopus (133) Google Scholar) and phosphoprotein Raf-1 (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). We have reported here for the first time that EGFR is likely to be a substrate of Cdc25A. This conclusion is based on the following findings. First, Cdc25A co-immunoprecipitated with EGFR in Cpd 5-treated Hep3B cells, and GST-Cdc25A directly bound to purified EGFR protein in vitro. Second, Cpd 5, a known inhibitor of Cdc25A phosphatase activity, induced a prolonged EGFR tyrosine phosphorylation. By contrast, in Hep3B cells expressing an inactivated Cdc25A, no EGFR tyrosine phosphorylation was found after the treatment with Cpd 5. This result is consistent with the report that the inactive mutant Cdc25A-C430S protein strongly interacts with both cyclin A-Cdk2 and cyclin E-Cdk2 but does not lead to an activation of Cdk2 kinase activity (38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Third, both purified GST-Cdc25A and endogenous Hep3B cellular Cdc25A dephosphorylated EGFR tyrosine phosphorylation in vitro and in cell culture; their dephosphorylation activity can be inhibited by Cpd 5, demonstrating that EGFR is a direct target of Cdc25A. Finally, in EGFR-transfected fibroblast NR-6 cells, Cdc25A was found to bind to wild-type and kinase-mutated EGFR but not to C-terminal-deleted EGFR. Furthermore, only wild-type EGFR was able to be tyrosine-phosphorylated with Cpd 5 treatment. These results suggest that Cdc25A binds to the C-terminal of EGFR and that EGFR kinase activity may be important to the action of Cdc25A on EGFR dephosphorylation It seems that EGFR dephosphorylation by Cdc25A is selective, because Cdc25A has no effect on insulin receptor tyrosine dephosphorylation. This selectivity is probably because of the structural differences between EGFR and insulin receptor. Most RTKs, such as EGFR and platelet-derived growth factor receptor, are monomers in the cell membrane. Ligand binding induces dimerization of these receptors (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). However, members of the insulin receptor family are disulfide-linked dimers of two polypeptide chains forming an α2β2 heterodimer (39Van Obberghen E. Diabetologia. 1994; 27: S125-S134Crossref Scopus (60) Google Scholar). Insulin binding to the extracellular domain of the insulin receptor induces a rearrangement in the quaternary heterotetrameric structure that leads to increased autophosphorylation of the cytoplasmic domain (40Hubbard S.R. Mohhammadi M. Schlessinger J. J. Biol. Chem. 1998; 273: 11987-11990Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). This structure may not be suitable for Cdc25A binding. The fact that the Cdc25A inhibitor Cpd 5 cannot induce insulin receptor tyrosine phosphorylation further supports our hypothesis. In the Cdc25 family, only Cdc25A showed marked tyrosine phosphatase activity directed against EGFR. Our results are consistent with those previous findings that Cdc25A is a more potent tyrosine phosphatase than Cdc25B or Cdc25C when Raf-1 and Cdk are used as the substrates (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar, 38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The mechanisms for this discrepancy have not yet been elucidated. One possibility is that the Cdc25 phosphatase activity is regulated by extensive phosphorylation of the N-terminal regulatory domain, since the alignment of all known dual-specific protein-tyrosine phosphatases shows that they are very different from each other in terms of length and amino acid sequence outside the putative catalytic domain (38Xu X. Burke S. J. Biol. Chem. 1996; 271: 5118-5124Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,41Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (335) Google Scholar). The Cdc25 protein phosphatase family has long been regarded as only a cell cycle regulator. However, recent studies have shown that Cdc25A may also play a central role in regulating mitogenic signal transduction pathways. For instance, it has been reported that Cdc25A can physically associate with Raf-1 and regulates Raf-1 tyrosine phosphorylation and its activity (26Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar, 27Xia K. Lee R.S. Narsimhan R.P. Mukhopadhyay N.K. Neel B.G. Roberts T.M. Mol. Cell. Biol. 1999; 19: 4819-4824Crossref PubMed Scopus (42) Google Scholar). The fact that Raf-1 kinase can form complexes with the cell cycle activator Cdc25A provides strong evidence that signal transduction pathways are linked with the cell cycle directly. In HeLa cells, overexpression of Cdc25A in whole cells induced ERK dephosphorylation, and the Cdc25A inhibitor Cpd 5 restored ERK phosphorylation and nuclear translocation, demonstrating that Cdc25A regulated endogenous ERK phosphorylation status in whole cells (34Vogt A. Adachi T. Ducruet A.P. Chesebrough J. Nemoto K. Carr B.I. Lazo J.S. J. Biol. Chem. 2001; 276: 20544-20550Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We also found that Cpd 5-inhibited Cdc25A activity could contribute to Cdk4 tyrosine phosphorylation and subsequent cell cycle block and cell growth inhibition (35Wang Z. Southwick E.C. Wang M. Kar S. Rosi K.S. Wilcox C.S. Lazo J.S. Carr B.I. Cancer Res. 2001; 61: 7211-7216PubMed Google Scholar). In this study, we again used Cpd 5 as a tool to reveal that Cdc25A was an EGFR phosphatase and that inhibition of Cdc25A activity by Cpd 5 caused prolonged EGFR tyrosine phosphorylation. These data suggest that Cdc25A acts as the phosphatase for several different substrates in the MAPK pathway and that Cpd 5-induced activation of the MAPK pathway can be triggered by either upstream activation of EGFR or Raf-1 or by direct inhibition of ERK phosphatase Cdc25A. Taken together, our data establish Cdc25A protein phosphatase as a key molecule in regulating and linking cell cycle progression and signal transduction pathway. Inhibition of Cdc25A activity by Cpd 5 not only overstimulates the MAPK pathway from membrane receptor to nuclear, which down-regulates gene transcription, but also directly inhibits cell cycle progression, eventually leading to growth inhibition and cell death. Thus, finding an efficient Cdc25A inhibitor may be of significant importance in controlling cancer cell growth and proliferation. We thank Dr. Sid Kar for helpful discussions.

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