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J. Biol. Chem., Vol. 277, Issue 22, 19470-19475, May 31, 2002

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

Ziqiu Wang, Meifang Wang, John S. Lazo, and Brian I. Carr^§

From the Thomas E. Starzl Transplantation Institute, Departments of Surgery and  Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

Received for publication, February 1, 2002, and in revised form, March 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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)¹ play an important role in the control of many fundamental cellular processes including cell cycle control, survival, metabolism, and differentiation (1). Tyrosine-phosphorylated growth factor receptors are rapidly inactivated by dephosphorylation, a process that is believed to negatively modulate signaling activity (2, 3). 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 (4, 5). 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 (6, 7). 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 (8-10). 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 (11, 12). 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 (13-16). Recent evidence suggests that Cdc25A may also have a role in the initiation of mitosis (17). 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 (18-24). 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 (25). 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 (26, 27). That Cdc25A is not highly promiscuous was suggested by the work of Zou et al. (28), which used a yeast two-hybrid system to screen >10⁶ clones and found Cdc25A bound only to 14-3-3, 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. 8).² 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 (29-31). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Human hepatoma cell line, Hep3B cells were maintained in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. The fibroblastic NR-6 cell line was a kind gift from Dr. Alan Wells (Dept. of Pathology, University of Pittsburgh). The wild-type NR-6 cells were EGFR-transfected 3T3 derivatives that lack endogenous EGFR. The EGFR mutants were constructed by standard methods as described previously (32). Briefly, the point mutant Met⁷²¹ was constructed by replacing Lys⁷²¹ with a methionine codon by site-directed mutagenesis. The mutant C1000 was also generated by site-directed mutagenesis in which stop codons were encoded after the amino acid number indicated. All NR-6 cells were grown in Eagle's minimum essential medium containing 10% fetal bovine serum and 350 µg/ml G418 (Invitrogen).

Immunoprecipitation and Western Blot Assay-- Hep3B cells were plated in 100-mm tissue culture dishes and treated with or without Cpd 5 for various times. After treatment, the cells were washed twice with cold phosphate-buffered saline and then lysed in 100 µl of immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin). Whole cell extracts (200 µg) were immunoprecipitated by anti-EGFR or anti-Cdc25A antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) with protein A-agarose (Sigma) overnight. The protein A-agarose pellets were washed three times with immunoprecipitation buffer and boiled in 40 µl of 2× sample buffer for 5 min. The proteins were resolved on a 10% SDS-polyacrylamide gel and transferred onto Hybond-polyvinylidene difluoride membranes (Amersham Biosciences). Membranes were blocked using Tris-buffered saline with Tween 20 (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 0.05% Tween 20) containing 1% bovine serum albumin for 1 h and then probed with anti-phosphotyrosine antibody (Oncogene Science, Cambridge, MA), anti-EGFR antibody, or anti-Cdc25A antibody (Santa Cruz Biotechnology) for 1 h. After washing four times with Tris-buffered saline with Tween 20, the membranes were probed with horseradish peroxidase-conjugated secondary antibody to allow detection of the appropriate bands using enhanced chemiluminescence (Amersham Biosciences).

GST Pull-down Assay-- GST pull-down experiments were performed as described previously, with some modifications (33). Briefly, the purified GST-Cdc25A fusion protein was incubated with 20 µl of glutathione-agarose beads equilibrated in 0.5× Superdex buffer (1× Superdex buffer: 25 mM HEPES, pH 7.5, 12.5 mM MgCl₂, 10 µM ZnSO₄, 150 mM KCl, 20% glycerol, 0.1% Nonidet P-40, and 1 mM EDTA) for 1-2 h at 4 °C and then washed with 0.5× Superdex buffer. EGFR protein (Promega Corp., Madison, WI) was then added to the washed beads and incubated overnight at 4 °C. The beads were washed again using 0.5× Superdex buffer, and the bound EGFR was eluted with SDS sample buffer and separated by SDS-PAGE.

EGFR Dephosphorylation Assay-- Activated (tyrosine-phosphorylated) EGFR protein was prepared by incubating Hep3B cells with 20 ng/ml EGF for 5 min. Whole cell lysates were immunoprecipitated with anti-EGFR antibody, and the EGFR immunoprecipitates were used as substrates for EGFR dephosphorylation assays. Equal amounts of EGFR immunoprecipitates were incubated with purified full-length GST-Cdc25A, GST-Cdc25B, or GST-Cdc25C proteins in 1× phosphatase buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol) for 15 min at 30 °C. The reaction was terminated by the addition of an equal volume of 2× sample buffer, and protein bands were separated by SDS-PAGE. The dephosphorylation of EGFR was examined by Western blot using anti-phosphotyrosine antibody (Oncogene Science). For EGFR dephosphorylation by Hep3B cell endogenous Cdc25A, EGF-stimulated Hep3B cell lysates were used as the substrate of endogenous Cdc25A phosphatase. A parallel group of Hep3B cells was treated with or without Cpd 5, and the non-denatured Hep3B cell lysates (non-denatured cell lysate buffer: 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM orthovanadate, 1 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), which had been precleared of EGFR, were immunoprecipitated with the anti-Cdc25A antibody. Equal amounts of EGF-stimulated Hep3B cell lysates were incubated with Hep3B cell-derived Cdc25A immunoprecipitates, which were washed twice with non-denatured cell lysate buffer and phosphatase buffer in 1× phosphatase buffer for 15 min at 30 °C. After removing Cdc25A immunoprecipitates, the dephosphorylated EGFR lysates were immunoprecipitated with anti-EGFR antibody and blotted with anti-phosphotyrosine antibody.

Cell Transfection-- The mammalian expression plasmid encoding the catalytically inactive C430S mutant of Cdc25A in a pcDNA3 vector was generously provided by Dr. Thomas Roberts (Dana Farber Cancer Institute, Boston, MA) (27). Transfections were carried out by the LipofectAMINE method following the manufacturer's instructions (Invitrogen). Briefly, Hep3B cells (100,000/well) were plated in 6-well plates and transfected with 1.0 µg/well plasmid cDNA in Opti-MEM transfection medium using the LipofectAMINE Plus reagent (Invitrogen). Five hours after transfection, the medium was replaced with complete growth medium, and the cells were allowed to recover for 48 h. Cells were treated with or without Cpd 5 (0-30 µM) for 60 min, and cell lysates were immunoprecipitated with anti-Cdc25A antibody and analyzed by Western blot using anti-phosphotyrosine and anti-EGFR antibodies.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cpd 5 Produces Persistent EGFR but Not Insulin Receptor Tyrosine Phosphorylation in Hep3B Cells-- We previously reported that Cpd 5 caused protein tyrosine phosphorylation in Hep3B hepatoma cells, which was associated with cell growth inhibition (9, 10). Furthermore, we found that, in rat hepatocytes, Cpd 5 produced persistent EGFR tyrosine phosphorylation and then triggered activation of the MAPK pathway (8). To investigate whether EGFR tyrosine phosphorylation by Cpd 5 is growth factor receptor-selective, we examined the effects of Cpd 5 on the insulin receptor in Hep3B cells. Hep3B cells were treated with Cpd 5 (20 µM from 5 min to 3 h). Whole cell lysates were immunoprecipitated with either anti-EGFR or anti-insulin receptor antibody and blotted with anti-phosphotyrosine antibody. Fig. 1A shows that Cpd 5 produced a prolonged EGFR tyrosine phosphorylation for at least 3 h, whereas no insulin receptor tyrosine phosphorylation was found even after we increased the concentration of Cpd 5 to 160 µM (data not shown). In Fig. 1B, we show that while insulin strongly induced insulin receptor tyrosine phosphorylation, EGF and Cpd 5 had almost no effect on insulin receptor tyrosine phosphorylation.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Cpd 5 induces EGFR but not insulin receptor tyrosine phosphorylation. A, Hep3B cells were treated with Cpd 5 (20 µM) from 0 to 180 min. Cell lysates were immunoprecipitated with anti-EGFR or anti-insulin receptor antibody and blotted with anti-phosphotyrosine antibody. The anti-EGFR immunoprecipitates were also blotted with anti-EGFR antibody to show the equal protein loading for EGFR tyrosine phosphorylation. B, Hep3B cells were treated with EGF (E) 10 ng/ml, Cpd 5 (C5) 20 µM, or insulin 1 µM for 30 min. Cell lysates were immunoprecipitated with anti-EGFR or anti-insulin receptor antibody and blotted with anti-phosphotyrosine antibody. The anti-EGFR or anti-insulin receptor immunoprecipitates were then blotted with anti-EGFR or anti-insulin receptor antibody as the protein loading control.

Interaction of Cdc25A with EGFR-- Based on our previous studies (9, 10), both EGF and Cpd 5 caused EGFR tyrosine phosphorylation, but the kinetics of EGFR phosphorylation are different. EGF is known to stimulate EGFR tyrosine kinase activity and induce EGFR tyrosine phosphorylation rapidly and transiently, whereas EGFR tyrosine phosphorylation after Cpd 5 treatment develops more slowly and is prolonged. Consequently, we hypothesized that EGFR hyperphosphorylation by Cpd 5 is probably due to its inhibitory effect on tyrosine phosphatase activity. However, we were unable to detect the prototype EGFR PTPase SH-PTP1 in Hep3B cells and found no inhibition of another prototype EGFR PTPase, SH-PTP2, by Cpd 5 (data not shown). Because we previously observed that Cdc25A activity was inhibited by vitamin K analogs in cell-free conditions and in cell cultures (31, 34, 35), we explored the possibility that EGFR hyperphosphorylation might be related to inhibition of Cdc25A activity. We first examined the physical association of EGFR with Cdc25A. Hep3B cells were treated with Cpd 5 (20 µM) from 0 to 60 min, and whole cell lysates were then immunoprecipitated with anti-Cdc25A antibody. The immunoprecipitates were resolved by SDS-PAGE and blotted with anti-phosphotyrosine, anti-EGFR, or anti-Cdc25A antibody, respectively. Fig. 2A shows that Cdc25A co-immunoprecipitated with EGFR in Hep3B cells. Furthermore, Cpd 5 treatment of the cells induced EGFR tyrosine phosphorylation in the EGFR-Cdc25A immunocomplexes in a concentration-dependent manner. To determine whether the binding of Cdc25A to EGFR is specific, we examined the physical association of Cdc25A with insulin receptor, a structurally different RTK, and found no co-immunoprecipitation (data not shown). These results suggested that Cdc25A selectively bound to and interacted with EGFR. To further test the Cdc25A-EGFR direct interaction, we examined the binding of GST-Cdc25A to EGFR using a GST pull-down assay (33). As shown in Fig. 2B, GST-Cdc25A bound to EGFR, whereas glutathione completely blocked this binding.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Cdc25A and EGFR interactions. A, Cdc25A co-immunoprecipitates with EGFR. Hep3B cells were treated with Cpd 5 (20 µM) from 0 to 60 min. The cell lysates were immunoprecipitated with anti-Cdc25A antibody and blotted with anti-phosphotyrosine, anti-EGFR, or anti-Cdc25A antibody, respectively. B, Cdc25A directly interacts with EGFR. Purified EGFR protein (200 ng) was incubated with either glutathione alone (1 nmol), GST-Cdc25A alone (1 µg), or glutathione and GST-Cdc25A. Bound EGFR protein was detected by Western blot analysis.

Inactivated Cdc25A Blocks Cpd 5-mediated EGFR Phosphorylation-- To further investigate the functional interaction of the putative Cdc25A and EGFR, we used a phosphatase-inactive Cdc25A mutant (C430S) to transfect Hep3B cells, because in many mammalian cell systems expression of a mutant enzyme can silence the endogenous active allele. Wild-type and C430S-expressing Hep3B cells were incubated with Cpd 5 (0-30 µM) for 1 h. Cell lysates were then immunoprecipitated with anti-Cdc25A antibody and immunoblotted with anti-phosphotyrosine, anti-EGFR, and anti-Cdc25A antibodies. As shown in Fig. 3, Cdc25A was co-immunoprecipitated with EGFR in both wild-type and C430S-expressing Hep3B cells. The physical association of Cdc25A and EGFR appeared to be somewhat weaker in C430S than in wild-type Hep3B cells, which might reflect the difference of binding activity between wild-type and mutant Cdc25A. Furthermore, Cpd 5 caused EGFR tyrosine phosphorylation in wild-type Hep3B cells but not in C430S-expressing Hep3B cells. These results indicate that Cpd 5 can inhibit Cdc25A activity and subsequently cause EGFR tyrosine phosphorylation.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Cpd 5-mediated EGFR tyrosine phosphorylation is blocked by the C430S mutant of Cdc25A. Wild-type and C430S-Cdc25A mutant-transfected Hep3B cells were treated with Cpd 5 (0, 20, and 30 µM) for 1 h. Cell lysates were immunoprecipitated with anti-Cdc25A antibody and blotted with anti-phosphotyrosine, anti-EGFR, or anti-Cdc25A antibody, respectively.

Recombinant GST-Cdc25A Dephosphorylates Tyrosine-phosphorylated EGFR in Vitro-- Because EGFR and Cdc25A proteins appeared to interact physically and functionally in cell cultures, we investigated whether cellular EGFR might act as a substrate for Cdc25A phosphatase. We examined the phosphatase action of recombinant GST-Cdc25A by incubating it with phosphorylated EGFR protein. Tyrosine-phosphorylated EGFR was purified from EGF-stimulated Hep3B cells after lysates were immunoprecipitated with anti-EGFR antibody. The EGFR immunocomplexes were then incubated with GST-Cdc25A in 1× phosphatase buffer at 30 °C for 15 min. After incubation, the EGFR immunocomplexes were washed with immunoprecipitation buffer and Western blotted using anti-phosphotyrosine antibody. We found that GST-Cdc25A dephosphorylated phospho-EGFR in a concentration-dependent manner. Furthermore, preincubation of 1 µg of GST-Cdc25A with Cpd 5 (0-40 µM) abrogated the dephosphorylation effect of GST-Cdc25A on phosphorylated EGFR (Fig. 4, A and B). In human cells, the Cdc25 family includes Cdc25A, Cdc25B, and Cdc25C. To examine whether only Cdc25A is the phosphatase for EGFR, we also treated phosphorylated EGFR with GST-Cdc25B or GST-Cdc25C. We found that GST-Cdc25C lacked any dephosphorylation activity, while GST-Cdc25B was markedly less active when compared with the GST-Cdc25A isoform (Fig. 4C). To further examine the Cdc25A substrate specificity, we incubated GST-Cdc25A with phosphorylated insulin receptor and found that insulin receptor also was not dephosphorylated by GST-Cdc25A (Fig. 4A).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   GST-Cdc25A dephosphorylates tyrosine-phosphorylated EGFR in vitro. A, tyrosine-phosphorylated EGFR or insulin receptor immunocomplexes, prepared from EGF or insulin-stimulated Hep3B cells (under "Experimental Procedures"), were incubated with recombinant GST-Cdc25A in 1× phosphatase buffer at 30 °C for 15 min. After washing with immunoprecipitation buffer, the EGFR or insulin receptor immunocomplexes were Western blotted and probed with anti-phosphotyrosine or anti-EGFR antibody. B, 1 µg of GST-Cdc25A was preincubated with Cpd 5 (0-40 µM) at 37 °C for 30 min, then incubated with tyrosine-phosphorylated EGFR immunocomplexes in 1× phosphatase buffer at 30 °C for 15 min. After washing with immunoprecipitation buffer, the EGFR immunocomplexes were blotted with anti-phosphotyrosine or anti-EGFR antibody. C, tyrosine-phosphorylated EGFR immunocomplexes were incubated with GST-Cdc25A, GST-Cdc25B, or GST-Cdc25C, respectively. This experimental procedure is the same as in A and B.

Hep3B Cellular Cdc25A Dephosphorylates Tyrosine-phosphorylated EGFR-- Because we found that GST-Cdc25A can dephosphorylate tyrosine-phosphorylated EGFR, we examined whether the endogenous cellular Cdc25A of Hep3B cells possessed this ability and whether Cpd 5 could inhibit its phosphatase activity. Hep3B cells were treated with Cpd 5 for 60 min at various concentrations (0-40 µM). Whole cell lysates were immunoprecipitated with anti-Cdc25A antibody, and the immunocomplex was incubated with either EGF or insulin-stimulated Hep3B cell lysates in phosphatase buffer at 30 °C for 15 min. After removing the Cdc25A immunocomplexes, the cell lysates were immunoprecipitated with either anti-EGFR or anti-insulin receptor antibody and analyzed by Western blot using anti-phosphotyrosine antibody. Fig. 5 shows that while untreated Hep3B cellular Cdc25A immunocomplexes dephosphorylated tyrosine-phosphorylated EGFR, Cpd 5 treatment inhibited this dephosphorylation activity in a concentration-dependent manner. By contrast, Cdc25A immunocomplexes did not dephosphorylate tyrosine-phosphorylated insulin receptor, and treatment of Cpd 5 did not alter insulin receptor phosphorylation status.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Cpd 5 inhibits Hep3B cellular Cdc25A phosphatase activity. Hep3B cells were treated with Cpd 5 for 60 min at various concentrations (0-40 µM). Whole cell lysates were immunoprecipitated with anti-Cdc25A antibody, and the immunocomplexes were incubated with EGF-stimulated or insulin-stimulated Hep3B cell lysates in 1× phosphatase buffer at 30 °C for 15 min. After removing the Cdc25A immunocomplexes, the cell lysates were immunoprecipitated with either anti-EGFR antibody or anti-insulin receptor antibody and blotted with anti-phosphotyrosine antibody. C is a control lysate that was not incubated with Cdc25A immunocomplexes.

Cdc25A Binds to the EGFR C-terminal-- To confirm that Hep3B cellular Cdc25A binds to and dephosphorylates tyrosine-phosphorylated EGFR, we used EGFR-transfected NR-6 fibroblastic cells to further investigate the EGFR-Cdc25A interaction. The parental NR-6 cells lack endogenous EGFR. Thus we used the transfected NR-6 cells with constructs containing wild-type EGFR, a point mutation (Fig. 6, M) within the full-length EGFR replacing the ATP-complexing lysine at amino acid 721 with methionine, or a C-terminal deletion (Fig. 6, C) at amino acid 1000 of EGFR (32). Cell lysates from each of these three NR-6 cell lines that had been treated with or without Cpd 5 were immunoprecipitated with anti-EGFR antibody and blotted with anti-phosphotyrosine, anti-EGFR, or anti-Cdc25A antibody, respectively. We found that Cpd 5 caused marked wild-type EGFR tyrosine phosphorylation and that EGFR was co-immunoprecipitated with Cdc25A in wild-type cells. In EGFR kinase-mutated NR-6 cells (M cells), although EGFR co-immunoprecipitated with Cdc25A, no EGFR tyrosine phosphorylation was caused by Cpd 5. In EGFR C-terminal-deleted NR-6 cells (C cells), neither EGFR-Cdc25A immunocomplexes nor EGFR tyrosine phosphorylation by Cpd 5 was found (Fig. 6A). Conversely, we also used anti-Cdc25A antibody to immunoprecipitate NR-6 cell lysates and blotted them with anti-EGFR and anti-Cdc25A antibodies. Fig. 6B shows that all wild-type, M, and C NR-6 cell lines contain Cdc25A protein, but no EGFR was found in EGFR C-terminal-deleted NR-6 cells.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Cdc25A and EGFR interaction in NR-6 fibroblastic cells. Wild-type EGFR (W), a point mutation within the full-length EGFR replacing the ATP-complexing lysine at amino acid 721 with methionine (M), or a C-terminal deletion at amino acid 1000 of EGFR (C) were expressed in NR-6 cells. Cell lysates from each of these three NR-6 cell lines that had been treated with or without Cpd 5 for 1 h were immunoprecipitated with anti-EGFR antibody (A) or anti-Cdc25A antibody (B) and blotted with anti-phosphotyrosine, anti-EGFR, or anti-Cdc25A antibody, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The activated and autophosphorylated growth factor receptors are subject to a rapid dephosphorylation by PTPases (36, 37). 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 (8). 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 (31, 35), 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 (17, 22). 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 (25) and phosphoprotein Raf-1 (26, 27). 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 (38). 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 (1). However, members of the insulin receptor family are disulfide-linked dimers of two polypeptide chains forming an ₂₂ heterodimer (39). 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 (40). 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 (26, 35, 38). 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 (38, 41).

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 (26, 27). 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 (34). We also found that Cpd 5-inhibited Cdc25A activity could contribute to Cdk4 tyrosine phosphorylation and subsequent cell cycle block and cell growth inhibition (35). 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.

	ACKNOWLEDGEMENT

We thank Dr. Sid Kar for helpful discussions.

	FOOTNOTES

^* This work is supported in part by National Institutes of Health Grants CA 82723 (to B. I. C.), CA 78039, and CA 52995 (J. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Thomas E. Starzl Transplantation Inst., Dept. of Surgery, University of Pittsburgh School of Medicine, E1552 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-624-6684; Fax: 412-624-6666; E-mail: carrbi@msx.upmc.edu.

Published, JBC Papers in Press, March 23, 2002, DOI 10.1074/jbc.M201097200

² Z. Wang and B. I. Carr, unpublished data.

	ABBREVIATIONS

The abbreviations used are: RTK(s), receptor-tyrosine kinase(s); PTPase(s), protein-tyrosine phosphatase(s); EGF, epidermal growth factor; EGFR, EGF receptor; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; ERK, extracellular signal-related kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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