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• W2102827209 abstract "Inhibition of general transcription and translation occurs during mitosis to preserve the high energy requirements needed for the dynamic structural changes that are occurring at this time of the cell cycle. Although the mitotic kinase Cdc2 appears to directly phosphorylate and inhibit key proteins directly involved in transcription and translation, the role of Cdc2 in regulating up-stream growth factor receptor-mediated signal transduction pathways is limited. In the present study, we examined mechanisms involved in uncoupling receptor-mediated activation of the extracellular signal-regulated (ERK) signaling pathway in mitotic cells. Treatment with epidermal growth factor (EGF) failed to activate the ERK pathway in mitotic cells, although partial activation of ERK could be achieved in mitotic cells treated with phorbol 12-myristate 13-acetate (PMA). The discrepancy between EGF and PMA-mediated ERK activation suggested that multiple events in the ERK pathway were regulated during mitosis. We show that Cdc2 inhibits EGF-mediated ERK activation through direct interaction and phosphorylation of several ERK pathway proteins, including the guanine nucleotide exchange factor, Sos-1, and Raf-1 kinase. Inhibition of Cdc2 activity with roscovitine in mitotic cells restored ERK activation by EGF and PMA. Similarly, mitotic inhibition of ERK activity in cells expressing active mutants of H-Ras and Raf-1 kinase could also be reversed following Cdc2 inhibition. In contrast, ERK activation in cells expressing active MEK1 was not inhibited during mitosis or affected by roscovitine. These data suggest that Cdc2 inhibits growth factor receptor-mediated ERK activation during mitosis by primarily targeting signaling proteins that are upstream of MEK1. Inhibition of general transcription and translation occurs during mitosis to preserve the high energy requirements needed for the dynamic structural changes that are occurring at this time of the cell cycle. Although the mitotic kinase Cdc2 appears to directly phosphorylate and inhibit key proteins directly involved in transcription and translation, the role of Cdc2 in regulating up-stream growth factor receptor-mediated signal transduction pathways is limited. In the present study, we examined mechanisms involved in uncoupling receptor-mediated activation of the extracellular signal-regulated (ERK) signaling pathway in mitotic cells. Treatment with epidermal growth factor (EGF) failed to activate the ERK pathway in mitotic cells, although partial activation of ERK could be achieved in mitotic cells treated with phorbol 12-myristate 13-acetate (PMA). The discrepancy between EGF and PMA-mediated ERK activation suggested that multiple events in the ERK pathway were regulated during mitosis. We show that Cdc2 inhibits EGF-mediated ERK activation through direct interaction and phosphorylation of several ERK pathway proteins, including the guanine nucleotide exchange factor, Sos-1, and Raf-1 kinase. Inhibition of Cdc2 activity with roscovitine in mitotic cells restored ERK activation by EGF and PMA. Similarly, mitotic inhibition of ERK activity in cells expressing active mutants of H-Ras and Raf-1 kinase could also be reversed following Cdc2 inhibition. In contrast, ERK activation in cells expressing active MEK1 was not inhibited during mitosis or affected by roscovitine. These data suggest that Cdc2 inhibits growth factor receptor-mediated ERK activation during mitosis by primarily targeting signaling proteins that are upstream of MEK1. Cdc2 is the major cyclin-dependent kinase that promotes cell cycle progression through the G2 phase and mitosis (1Norbury C. Nurse P. Ciba Found. Symp. 1990; 150 (177–183): 168-177PubMed Google Scholar). Through direct phosphorylation, Cdc2 regulates important mitotic structural changes including nuclear envelope breakdown (2Luscher B. Brizuela L. Beach D. Eisenman R.N. EMBO J. 1991; 10: 865-875Crossref PubMed Scopus (73) Google Scholar, 3Nigg E.A. Semin. Cell Biol. 1992; 3: 245-253Crossref PubMed Scopus (64) Google Scholar), centrosome assembly (4Nigg E.A. Blangy A. Lane H.A. Exp. Cell Res. 1996; 229: 174-180Crossref PubMed Scopus (72) Google Scholar), and Golgi complex fragmentation (5Lowe M. Rabouille C. Nakamura N. Watson R. Jackman M. Jamsa E. Rahman D. Pappin D.J. Warren G. Cell. 1998; 94: 783-793Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). In addition, Cdc2 also functions to inhibit other cellular processes that would utilize energy stores needed for the dynamic structural changes that are occurring at this time of the cell cycle. For example, the role of Cdc2 is well established in inhibiting transcription during mitosis (6Hartl P. Gottesfeld J. Forbes D.J. J. Cell Biol. 1993; 120: 613-624Crossref PubMed Scopus (82) Google Scholar) through direct phosphorylation of proteins such as RNA polymerase II and TFIIIB (7Gottesfeld J.M. Wolf V.J. Dang T. Forbes D.J. Hartl P. Science. 1994; 263: 81-84Crossref PubMed Scopus (109) Google Scholar, 8Leresche A. Wolf V.J. Gottesfeld J.M. Exp. Cell Res. 1996; 229: 282-288Crossref PubMed Scopus (67) Google Scholar, 9Gebara M.M. Sayre M.H. Corden J.L. J. Cell Biochem. 1997; 64: 390-402Crossref PubMed Scopus (69) Google Scholar). Moreover, evidence suggests that Cdc2 may indirectly cause inhibition of transcription during mitosis by targeting other unknown kinases (6Hartl P. Gottesfeld J. Forbes D.J. J. Cell Biol. 1993; 120: 613-624Crossref PubMed Scopus (82) Google Scholar). Other metabolic processes such as the high energy requirements of ribosome biogenesis and protein translation are also inhibited during mitosis through Cdc2-mediated phosphorylation (10Heix J. Vente A. Voit R. Budde A. Michaelidis T.M. Grummt I. EMBO J. 1998; 17: 7373-7381Crossref PubMed Scopus (133) Google Scholar, 11Sirri V. Hernandez-Verdun D. Roussel P. J. Cell Biol. 2002; 156: 969-981Crossref PubMed Scopus (123) Google Scholar). Recently, Cdc2 was shown to inhibit the p90 S6 ribosomal kinase-1 through direct phosphorylation providing an additional level of regulation of mitotic inhibition of translation (12Shah O.J. Ghosh S. Hunter T. J. Biol. Chem. 2003; 278: 16433-16442Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Thus, mitotic cells coordinate the inhibition of biological processes that might interfere with the energetically demanding structural changes that are necessary to ensure the equal segregation of genetic material into each daughter cell. Mitotic cells are also less responsive to extracellular growth factor stimulation as compared with interphase cells. For example, epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein or extracellular signal-regulated kinases; PMA, phorbol 12-myristate 13-acetate. receptor activity is reduced during mitosis following stimulation, reportedly because of reduced EGF binding and inhibition of EGF receptor dimerization (13Kiyokawa N. Lee E.K. Karunagaran D. Lin S.Y. Hung M.C. J. Biol. Chem. 1997; 272: 18656-18665Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Klein S. Kaszkin M. Barth H. Kinzel V. Biochem. J. 1997; 322: 937-946Crossref PubMed Scopus (11) Google Scholar, 15Newberry E.P. Pike L.J. Biochem. Biophys. Res. Commun. 1995; 208: 253-259Crossref PubMed Scopus (15) Google Scholar), possibly through a mechanism involving direct phosphorylation by Cdc2 (16Kiyokawa N. Karunagaran D. Lee E.K. Xie Y. Yan D.H. Hung M.C. Oncogene. 1997; 15: 2633-2641Crossref PubMed Scopus (8) Google Scholar). Inhibition of EGF receptor activity would also be beneficial for preventing the activation of signal transduction pathways that promote gene expression to preserve energy needs that are required for mitotic structural changes. The ability for EGF to activate the extracellular signal-regulated kinase (ERK) signal transduction pathway has been shown to be inhibited in mitotic cells as compared with interphase cells (17Gomez-Cambronero J. FEBS Lett. 1999; 443: 126-130Crossref PubMed Scopus (16) Google Scholar). EGF receptor-mediated activation of the ERK proteins occurs sequentially through Ras G-proteins, Raf kinases, and direct phosphorylation by the mitogen activated protein (MAP) or ERK kinases (MEKs) (18Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 19Shapiro P. Crit. Rev. Clin. Lab. Sci. 2002; 39: 285-330Crossref PubMed Scopus (86) Google Scholar). The coupling between the EGF receptor and the Ras G-protein occurs primarily through the Src homology 2 domain containing adaptor proteins Shc and Grb2 interacting with phosphorylated tyrosines on the EGF receptor and GTP loading on Ras by the guanine nucleotide exchange factor Sos-1 (20Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell Biol. 2001; 2: 127-137Crossref PubMed Scopus (5670) Google Scholar). It is not known whether Cdc2 inhibits EGF receptor signaling during mitosis by targeting these proteins that link the activated EGR receptor with ERK activation. ERK pathway proteins may undergo unique regulation during mitosis. Recently phosphorylated MEK-1 has been reported to undergo partial proteolysis at the N terminus during mitosis, which results in the inability for MEK1 to interact with and activate ERK1/2 proteins (21Harding A. Giles N. Burgess A. Hancock J.F. Gabrielli B.G. J. Biol. Chem. 2003; 278: 16747-16754Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, it is unlikely that MEK1 is completely uncoupled from ERK1/2 during mitosis because activation of protein kinase C by treatment with phorbol esters can still activate the Raf-1/MEK/ERK pathway in mitotic cells (14Klein S. Kaszkin M. Barth H. Kinzel V. Biochem. J. 1997; 322: 937-946Crossref PubMed Scopus (11) Google Scholar). Moreover, we have reported that the observed proteolysis of MEK1 during mitosis may not be because of N-terminal cleavage but is the result of cross-reactivity of phosphorylation-specific MEK1/2 antibodies used in these studies with nucleophosmin, an abundant nucleolar protein of ∼33 kDa that is phosphorylated by Cdc2 during mitosis (22Cha H. Hancock C. Dangi S. Maiguel D. Carrier F. Shapiro P. Biochem. J. 2004; 378: 857-865Crossref PubMed Scopus (48) Google Scholar). The functions of the ERK pathway and mechanisms of regulation during G2 or M phase progression are not well defined and somewhat controversial. Earlier studies that suggested a potential involvement of ERK in mitotic events reported that Raf-1 was activated in cells arrested in mitosis with nocodazole (23Laird A.D. Taylor S.J. Oberst M. Shalloway D. J. Biol. Chem. 1995; 270: 26742-26745Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, Raf-1 activation in nocodazole-induced mitotic cells appeared to occur through a mechanism that is independent of receptor-mediated Ras and did not result in corresponding downstream MEK1 protein activation (24Ziogas A. Lorenz I.C. Moelling K. Radziwill G. J. Biol. Chem. 1998; 273: 24108-24114Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Laird A.D. Morrison D.K. Shalloway D. J. Biol. Chem. 1999; 274: 4430-4439Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Similarly, although tyrosine phosphorylation of Raf-1 by Src kinase appears to be partially responsible for mitotic Raf-1 activity (24Ziogas A. Lorenz I.C. Moelling K. Radziwill G. J. Biol. Chem. 1998; 273: 24108-24114Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), a comprehensive analysis of the phosphorylation-dependent mechanisms that regulate mitotic Raf-1 activity has not been done. Thus, the function and targets of active Raf-1 during mitosis remain unknown. Nonetheless, a requirement for MEK1/2 and ERK1/2 activity during G2 phase and mitosis has been suggested in cell cultures using pharmacological inhibitors of MEK1/2 (26Hayne C. Tzivion G. Luo Z. J. Biol. Chem. 2000; 275: 31876-31882Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 27Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (124) Google Scholar), dominant negative MEK1 mutants (28Wright J.H. Munar E. Jameson D.R. Andreassen P.R. Margolis R.L. Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11335-11340Crossref PubMed Scopus (159) Google Scholar), MEK2 knock-out cell lines (29Abbott D.W. Holt J.T. J. Biol. Chem. 1999; 274: 2732-2742Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), or Raf-1/MEK1/2/ERK1/2 down-regulation using RNA interference (30Liu X. Yan S. Zhou T. Terada Y. Erikson R.L. Oncogene. 2004; 23: 763-776Crossref PubMed Scopus (136) Google Scholar). Phosphorylated MEK1/2 and ERK1/2 proteins have been shown to localize to the centrosomes and kinetochores, respectively, of mitotic cells and may regulate the function of proteins involved in metaphase to anaphase transitions (31Shapiro P.S. Vaisberg E. Hunt A.J. Tolwinski N.S. Whalen A.M. McIntosh J.R. Ahn N.G. J. Cell Biol. 1998; 142: 1533-1545Crossref PubMed Scopus (194) Google Scholar, 32Zecevic M. Catling A.D. Eblen S.T. Renzi L. Hittle J.C. Yen T.J. Gorbsky G.J. Weber M.J. J. Cell Biol. 1998; 142: 1547-1558Crossref PubMed Scopus (194) Google Scholar). Raf-1 and MEK1 have also been implicated in regulating Golgi complex fragmentation, which occurs as cells enter mitosis (33Acharya U. Mallabiabarrena A. Acharya J.K. Malhotra V. Cell. 1998; 92: 183-192Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 34Colanzi A. Deerinck T.J. Ellisman M.H. Malhotra V. J. Cell Biol. 2000; 149: 331-339Crossref PubMed Scopus (88) Google Scholar, 35Colanzi A. Sutterlin C. Malhotra V. J. Cell Biol. 2003; 161: 27-32Crossref PubMed Scopus (59) Google Scholar, 36Xie S. Wang Q. Ruan Q. Liu T. Jhanwar-Uniyal M. Guan K. Dai W. Oncogene. 2004; 23: 3822-3829Crossref PubMed Scopus (39) Google Scholar). Although ERK1/2 proteins are the only known substrates for MEK1 and MEK2, a role for the ERKs in regulating MEK1-induced mitotic Golgi fragmentation was not demonstrated in these studies. The aforementioned studies support a model where non-extracellular signal-mediated ERK pathway activation or phosphorylation occurs in localized intracellular regions and is important for mitotic transitions. The present study focused on identifying mechanisms involved in the inhibition of receptor-mediated ERK signaling in mitotic cells. Our findings indicate that Cdc2 plays a key role in negative regulation of receptor-mediated ERK signal transduction by forming complexes with several proteins that couple the EGF receptor with ERK activation. Thus, in addition to directly inhibiting proteins involved in transcription and translation, Cdc2 serves an important role in inhibiting extracellular growth factor-regulated signaling pathways during mitosis. Cell Culture and Reagents—HeLa or MDA-MB-468 cells were cultured in a complete medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (penicillin, 100 units/ml, and streptomycin, 100 μg/ml) from Intergen Life Sciences (Carlsbad, CA). EGF, LY294002, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma and used at final concentrations of 100 ng/ml, 25 μm, and 0.1 μm, respectively. The MEK1/2 inhibitor, U0126, was purchased from Calbiochem (La Jolla, CA) and used at a final concentration of 10 μm. AG1517, roscovitine, and staurosporine were purchased from Calbiochem and used at final concentrations of 500 nm, 60 μm, and 2 μm, respectively. Antibodies specific for cyclin B1 (sc-245), ERK2 (sc-154), Sos-1 (sc-256), Grb2 (sc-255), Raf-1 (sc-133), and Cdc2 (sc-54) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for phosphorylated ERK1/2 (M-8159), α-tubulin (T-6557), and β-actin (A-1978) were purchased from Sigma. The antibodies specific for phosphorylated MEK1/2 (number 9121) and pY(1068)EGFR (number 2236S) were purchased from Cell Signaling Technologies (Beverly, MA). Peroxidase-conjugated mouse and rabbit secondary antibodies were purchased from KPL (Gaithersburg, MD). Cell Synchronization—HeLa cells were synchronized as has been previously described (40Shah B.H. Catt K.J. Mol. Pharmacol. 2002; 61: 343-351Crossref PubMed Scopus (81) Google Scholar, 41Tebar F. Llado A. Enrich C. FEBS Lett. 2002; 517: 206-210Crossref PubMed Scopus (38) Google Scholar). Briefly, cells (50% confluent) were treated with 2 mm thymidine in Dulbecco's modified Eagle's medium + fetal bovine serum for about 16 h. Cells were released back into the cell cycle by washing with Hanks' buffered saline solution and incubated for 8 h in Dulbecco's modified Eagle's medium + fetal bovine serum in the absence of thymidine. Cells were then treated a second time with 2 mm thymidine in Dulbecco's modified Eagle's medium + fetal bovine serum for 16 h, which results in greater than 80% of the cells synchronized in the G1 phase of the cell cycle. Synchronized cells were washed with Hanks' buffered saline solution and then released back into the cell cycle and harvested at various times under various treatments. Cells were arrested in prometaphase of mitosis by treating with nocodazole (100 ng/ml), vinblastine (10 μg/ml), or paclitaxel (1 μm), all purchased from Sigma, for 12–14 h. In some cases, cells transfected with constitutively active mutants of MEK1 (kindly provided by Dr. Natalie Ahn, University of Colorado), BXB-Raf-1 (kindly provided by Dr. Ulf Rapp, University of Wurzburg), of V12 H-Ras (kindly provided by Dr. Melanie Cobb, University of Texas-Southwestern)) were arrested with nocodazole and treated in the presence or absence of roscovitine during the last hour of nocodazole treatment. Mitotic Shakeoff Analysis—Cells were synchronized at the G1/S phase boundary using excess thymidine and then released back into the cell cycle for 9 h, where the peak number of mitotic cells can be obtained (27Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (124) Google Scholar, 37Cha H. Shapiro P. J. Cell Biol. 2001; 153: 1355-1368Crossref PubMed Scopus (66) Google Scholar). Mitotic cells were dislodged by gently tapping the plates and placed in sterile tubes containing complete medium. The remaining attached cells (G2 phase) and the mitotic cells were immediately treated with EGF or PMA for 5 or 30 min followed by harvesting and immunoblot analysis. Immunoblotting—Immunoblotting was done as has been previously described. Briefly, protein lysates were collected from synchronized cells that were washed twice with cold phosphate-buffered saline, lysed with 300 μl of cold tissue lysis buffer (20 mm Tris, pH 7.4, 137 mm NaCl, 2mm EDTA, 1% Triton X-100, 25 mm β-glycerophosphate, 2 mm sodium pyrophosphate, 10% glycerol, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine), allowed to incubate on ice for about 10 min, and then centrifuged at 14,000 revolutions per minute to clarify the lysates of insoluble material. The lysates were then diluted with an equal volume of 2× SDS-sample buffer before resolving them by SDS-PAGE, prior to immunoblot analysis. Immunoblots were detected by enhanced chemiluminescence Western blotting reagents (Amersham Biosciences). Co-immunoprecipitations—Protein interaction studies were examined in co-immunoprecipitation assays using lysates harvested from asynchronous and mitotic cells stimulated with or without EGF or PMA. Lysates (200–300 μg) were incubated with 2 μl of the indicated primary antibody (0.1–0.2 mg/ml) for 2 h on ice, followed by incubation with 25 μl of a 50% slurry of protein-Sepharose A/G 4 Fast Flow (Amersham Biosciences) at 4 °C for 4 h under constant mixing. The immune complexes were washed twice (0.5 ml each) with 25 mm Hepes, pH 7.4, 25 mm MgCl2, and 1 mm dithiothreitol or with tissue lysis buffer before reconstitution in SDS-PAGE sample buffer and immunoblot analysis of immunoprecipitated and co-immunoprecipitating proteins. Densitometry Analysis—The relative amount of immunoprecipitated protein was measured by densitometry analysis of the immunoblots using NIH imager software. The relative density of the protein bands was calculated in the area encompassing the immunoreactive protein band and subtracting the background of an adjacent non-reactive area in the same lane of the protein of interest. To account for any variations in the amount of protein that was immunoprecipitated, the ratio of the co-immunoprecipitated protein to immunoprecipitated protein was calculated. ERK Activity in Mitotic Cells Treated with EGF or PMA— Previously, it has been shown that activation of the EGF receptor is inhibited during mitosis (13Kiyokawa N. Lee E.K. Karunagaran D. Lin S.Y. Hung M.C. J. Biol. Chem. 1997; 272: 18656-18665Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Klein S. Kaszkin M. Barth H. Kinzel V. Biochem. J. 1997; 322: 937-946Crossref PubMed Scopus (11) Google Scholar, 15Newberry E.P. Pike L.J. Biochem. Biophys. Res. Commun. 1995; 208: 253-259Crossref PubMed Scopus (15) Google Scholar, 17Gomez-Cambronero J. FEBS Lett. 1999; 443: 126-130Crossref PubMed Scopus (16) Google Scholar). Similarly, EGF receptor-mediated activation of the ERK pathway is inhibited in mitotic cells (17Gomez-Cambronero J. FEBS Lett. 1999; 443: 126-130Crossref PubMed Scopus (16) Google Scholar). In contrast, phorbol ester induction of protein kinase C-mediated signaling pathways has been suggested to be intact in mitotic cells and result in a G2-arrest (14Klein S. Kaszkin M. Barth H. Kinzel V. Biochem. J. 1997; 322: 937-946Crossref PubMed Scopus (11) Google Scholar, 38Kosaka C. Sasaguri T. Ishida A. Ogata J. Am. J. Physiol. 1996; 270: C170-C178Crossref PubMed Google Scholar, 39Kaszkin M. Furstenberger G. Richards J. Seidler L. Kinzel V. Cancer Res. 1991; 51: 4328-4335PubMed Google Scholar). To explore the mechanisms involved in inactivation of receptor-mediated signaling in mitotic cells, the activation of ERK was compared in cells arrested in mitosis by nocodazole treatment or asynchronous cells following treatment with EGF or PMA. In contrast to previous studies, which claimed that PMA-induced signaling was largely independent of the cell cycle (14Klein S. Kaszkin M. Barth H. Kinzel V. Biochem. J. 1997; 322: 937-946Crossref PubMed Scopus (11) Google Scholar); in our hands PMA-treated cells showed a delayed ERK activation in mitotic cells compared with asynchronous cells (Fig. 1A). As expected, ERK activation in response to EGF was almost completely inhibited in mitotic cells (Fig. 1B). Increased cyclin B1 expression (Fig. 1A) as well as analysis of mitotic chromosomes and fluorescence-activated cell sorting confirmed that nocodazole-treated cells were arrested in mitosis (data not shown). The effect of EGF activation of ERK was also tested in the context of other pharmacological agents that induce mitotic arrest. As shown, EGF stimulation of ERK was inhibited in cells arrested in mitosis using vinblastine (Fig. 2A) or paclitaxel (Fig. 2, B and C). Similar to nocodazole-arrested cells, the PMA-mediated activation of ERK was delayed in cells arrested in mitosis with vinblastine or paclitaxel (Fig. 2, A and C). We have also demonstrated that mitotic cells isolated by gently dislodging (shake off) from synchronized cells showed a similar decreased ERK activation in response to EGF or PMA as compared with the remaining adherent cells (data not shown). These data indicate that mitotic inhibition of ERK signaling is not related to the pharmacological agents used to induce mitosis. The differences in ERK activation observed following PMA or EGF treatment in mitotic cells prompted us to examine mechanisms responsible for PMA-mediated ERK activity and down-regulation of growth factor receptor-mediated ERK signaling at this time of the cell cycle. PMA-induced ERK Activation Does Not Require EGF Receptor Tyrosine Kinase Activity—PMA-induced protein kinase C activation has been linked to transactivation of the EGF receptor (40Shah B.H. Catt K.J. Mol. Pharmacol. 2002; 61: 343-351Crossref PubMed Scopus (81) Google Scholar, 41Tebar F. Llado A. Enrich C. FEBS Lett. 2002; 517: 206-210Crossref PubMed Scopus (38) Google Scholar, 42Chen N. Ma W.Y. She Q.B. Wu E. Liu G. Bode A.M. Dong Z. J. Biol. Chem. 2001; 276: 46722-46728Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To determine whether the PMA-induced ERK activation observed during mitosis involves the EGF receptor, asynchronous or nocodazole-arrested cells were pretreated with AG1517, which is a potent and specific inhibitor of EGF receptor tyrosine kinase activity (43Fry D.W. Kraker A.J. McMichael A. Ambroso L.A. Nelson J.M. Leopold W.R. Connors R.W. Bridges A.J. Science. 1994; 265: 1093-1095Crossref PubMed Scopus (815) Google Scholar). Cells were then stimulated with either PMA or EGF and analyzed for ERK activation by immunoblotting. AG1517 had no effect on PMA-induced ERK activation in mitotic or asynchronous cells (Fig. 3A). As a control, pre-treatment with AG1517 effectively blocked EGF activation of ERK in asynchronous cells (Fig. 3B). Thus, PMA-induced activation of ERK during mitosis in HeLa cells does not appear to involve transactivation of the EGF receptor. Changes in Sos-1, Raf-1, and EGF Receptor Phosphorylation during Mitosis—We next examined the phosphorylation status of the guanine nucleotide exchange factor Sos-1 and the Raf-1 kinase during mitosis to determine whether this post-translational modification could account for the inhibition of EGF or PMA-mediated ERK activation. Phosphorylation decreases the mobility of Sos-1 and Raf-1 in a polyacrylamide gel and this can be easily examined by immunoblot analysis. First, Sos-1, which is important for activating Ras, and the Ras effector kinase Raf-1 were examined. Sos-1 has been shown to be phosphorylated by MAP kinases following EGF receptor activation and this phosphorylation is thought to act as a negative feedback mechanism by inhibiting interactions with the adaptor proteins such as Grb2 resulting in the uncoupling of receptor-mediated signaling (44Corbalan-Garcia S. Degenhardt K.R. Bar-Sagi D. Oncogene. 1996; 12: 1063-1068PubMed Google Scholar, 45Dong C. Waters S.B. Holt K.H. Pessin J.E. J. Biol. Chem. 1996; 271: 6328-6332Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Previous studies demonstrate that Raf-1 is hyper-phosphorylated during mitosis (25Laird A.D. Morrison D.K. Shalloway D. J. Biol. Chem. 1999; 274: 4430-4439Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), although the functions of these phosphorylations are not known. Treatment of cells with EGF or PMA in asynchronous cells induced a slower migrating gel-retarded form of Sos-1 and Raf-1 indicative of phosphorylation (Fig. 4A). Moreover, AG1517 blocked EGF-induced phosphorylation of Sos-1 and Raf-1 but had no effect on PMA-induced Sos-1 or Raf-1 phosphorylation in asynchronous cells (Fig. 4A). These data further demonstrate the specificity of AG1517 in targeting the EGF receptor. In contrast, gel mobility of Sos-1 and Raf-1 was retarded to a greater extent in cells arrested in mitosis with nocodazole (Fig. 4A). Treatment of mitotic cells with EGF, PMA, or AG1517 had no effect on Sos-1 or Raf-1 gel mobility (Fig. 4A). These data demonstrate that Sos-1 and Raf-1 are hyperphosphorylated during mitosis through a mechanism involving mitotic specific kinases. To examine potential classes of kinases that may be responsible for hyperphosphorylated Sos-1 and Raf-1 during mitosis, cells arrested in mitosis with nocodazole were treated during the last 1 h prior to harvesting with the Cdc2 inhibitor roscovitine, the general kinase inhibitor staurosporine, the PI3K inhibitor LY294002, or the MEK1/2 inhibitor U0126. Both roscovitine and staurosporine inhibited the hyperphosphorylated forms of Sos-1 and Raf-1 as suggested by the increase in gel mobility (Fig. 4B). The decrease in Sos-1 phosphorylation was similar when using roscovitine or staurosporine indicating that Cdc2 was the primary kinase that directly or indirectly caused phosphorylation of Sos-1 during mitosis (Fig. 4B). In contrast, greater inhibition of Raf-1 gel mobility occurred in cells treated with staurosporine compared with roscovitine (Fig. 4B). This supports previous studies that suggest Cdc2 and other kinases directly or indirectly mediate Raf-1 phosphorylation during mitosis (25Laird A.D. Morrison D.K. Shalloway D. J. Biol. Chem. 1999; 274: 4430-4439Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Cells treated with U0126 or LY294002 had little effect on the mitotic Raf-1 gel mobility indicating that MEK1/2 and PI3K-mediated signaling does not affect Raf-1 phosphorylation events during mitosis (Fig. 4B). The previous data suggested that Cdc2 might regulate multiple proteins involved in the activation of the ERK pathway. Previously, it was suggested that ligand-induced autophosphorylation of tyrosine residues within the cytoplasmic domain of the EGF receptor is inhibited in mitotic cells (13Kiyokawa N. Lee E.K. Karunagaran D. Lin S.Y. Hung M.C. J. Biol. Chem. 1997; 272: 18656-18665Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Klein S. Kaszkin M. Barth H. Kinzel V. Biochem. J. 1997; 322: 937-946Crossref PubMed Scopus (11) Google Scholar, 15Newberry E.P. Pike L.J. Biochem. Biophys. Res. Commun. 1995; 208: 253-259Crossref PubMed Scopus (15) Google Scholar, 46Kinzel V. Kaszkin M. Blume A. Richards J. Cancer Res. 1990; 50: 7932-7936PubMed Google Scholar). However, the specific tyrosine residues that may have reduced phosphorylation in response to EGF have not been defined. To extend our examinatio" @default.
• W2102827209 created "2016-06-24" @default.
• W2102827209 creator A5016466331 @default.
• W2102827209 creator A5073626775 @default.
• W2102827209 date "2005-07-01" @default.
• W2102827209 modified "2023-10-14" @default.
• W2102827209 title "Cdc2-mediated Inhibition of Epidermal Growth Factor Activation of the Extracellular Signal-regulated Kinase Pathway during Mitosis" @default.
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