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• W1976335054 abstract "Activation of the mitogen-activated protein kinase (MAPK) pathway by growth factors or phorbol esters during G2 phase delays entry into mitosis; however, the role of the MAPK pathway during G2/M progression remains controversial. Here, we demonstrate that activation of the MAPK pathway with either epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate induces a G2 phase delay independent of known G2 phase checkpoint pathways but was specifically dependent on MAPK/extracellular signal-regulated kinase kinase (MEK1). Activation of MAPK signaling also blocked exit from a G2 phase checkpoint arrest. Both the G2 phase delay and blocked exit from the G2 checkpoint arrest were mediated by the MEK1-dependent destabilization of the critical G2/M regulator cdc25B. Reintroduction of cdc25B overcame the MEK1-dependent G2 phase delay. Thus, we have demonstrated a new function for MEK1 that controls G2/M progression by regulating the stability of cdc25B. This represents a novel mechanism by which factors that activate MAPK signaling can influence the timing of entry into mitosis, particularly exit from a G2 phase checkpoint arrest. Activation of the mitogen-activated protein kinase (MAPK) pathway by growth factors or phorbol esters during G2 phase delays entry into mitosis; however, the role of the MAPK pathway during G2/M progression remains controversial. Here, we demonstrate that activation of the MAPK pathway with either epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate induces a G2 phase delay independent of known G2 phase checkpoint pathways but was specifically dependent on MAPK/extracellular signal-regulated kinase kinase (MEK1). Activation of MAPK signaling also blocked exit from a G2 phase checkpoint arrest. Both the G2 phase delay and blocked exit from the G2 checkpoint arrest were mediated by the MEK1-dependent destabilization of the critical G2/M regulator cdc25B. Reintroduction of cdc25B overcame the MEK1-dependent G2 phase delay. Thus, we have demonstrated a new function for MEK1 that controls G2/M progression by regulating the stability of cdc25B. This represents a novel mechanism by which factors that activate MAPK signaling can influence the timing of entry into mitosis, particularly exit from a G2 phase checkpoint arrest. IntroductionThe canonical MAPK 5The abbreviations used are: MAPKmitogen-activated protein kinasecdkcyclin-dependent kinaseEGFepidermal growth factorERKextracellular signal-regulated kinaseGFPgreen fluorescent proteinMEFmouse embryonic fibroblastMEKMAPK/ERK kinasePKCprotein kinase CsiRNAsmall interfering RNATPA12-O-tetradecanoylphorbol-13-acetateATMataxia telangiectasia mutatedATRATM-Rad3 related. pathway of Ras, Raf, MEK, and ERK provides a sensitive mechanism for transducing extracellular signals critical for cell growth and development (1.Harding A. Tian T. Westbury E. Frische E. Hancock J.F. Curr. Biol. 2005; 15: 869-873Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). ERK-dependent phosphorylation of a broad range of substrates is the primary signaling output of the pathway (2.Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2248) Google Scholar). The ability of the MAPK pathway to influence entry into the cell cycle has been well established (3.Edelmann H.M. Kühne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4.Lavoie J.N. L'Allemain G. Brunet A. Müller R. Pouysségur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 5.Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (122) Google Scholar). Signaling through this pathway also has a role in G2 phase (6.Wright 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 (157) Google Scholar, 7.Liu X. Yan S. Zhou T. Terada Y. Erikson R.L. Oncogene. 2004; 23: 763-776Crossref PubMed Scopus (132) Google Scholar, 8.Shinohara M. Mikhailov A.V. Aguirre-Ghiso J.A. Rieder C.L. Mol. Biol. Cell. 2006; 17: 5227-5240Crossref PubMed Scopus (28) Google Scholar), possibly to regulate Golgi disassembly (9.Colanzi A. Sutterlin C. Malhotra V. J. Cell Biol. 2003; 161: 27-32Crossref PubMed Scopus (58) Google Scholar).Growth factors activate the canonical MAPK pathway through specific receptors, whereas the tumor-promoting phorbol esters activate this pathway via PKC (10.El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). When either growth factors or phorbol esters are added in G2 phase they can induce a G2 phase cell cycle delay (11.Barth H. Kinzel V. Exp. Cell Res. 1994; 212: 383-388Crossref PubMed Scopus (23) Google Scholar, 12.Barth H. Kinzel V. J. Cell. Physiol. 1995; 162: 44-51Crossref PubMed Scopus (10) Google Scholar); however, the exact mechanism of the delay is poorly understood. Activation of the MAPK pathway by growth factors and phorbol esters has been implicated in the G2 phase delay (13.Dangi S. Chen F.M. Shapiro P. Cell Prolif. 2006; 39: 261-279Crossref PubMed Scopus (33) Google Scholar), with the ERK-dependent up-regulation of the cdk inhibitor p21WAF1 identified as a key component of the delay (13.Dangi S. Chen F.M. Shapiro P. Cell Prolif. 2006; 39: 261-279Crossref PubMed Scopus (33) Google Scholar). Other mechanisms, including increased protein phosphatase 2A activity, have also been proposed (14.Klingler-Hoffmann M. Barth H. Richards J. König N. Kinzel V. Eur. J. Cell Biol. 2005; 84: 719-732Crossref PubMed Scopus (8) Google Scholar).Cells respond to a variety of stresses by imposing a G2 phase delay through the action of cell cycle checkpoint mechanisms. The checkpoint mechanisms impose the arrest by blocking activation of cyclin B/cdk1, the driver of mitosis. Major checkpoint mechanisms demonstrated to impose a G2 phase arrest in response to DNA damage are the ATM and related ATR-dependent pathways. ATM and ATR are the apical components of pathways that signal through Chk1 and Chk2 to block cdc25-dependent activation of the mitotic cyclin/cdks (15.O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The p38MAPK-MAPKAPK2 pathway is involved in a separate G2 arrest pathway (16.Bulavin D.V. Amundson S.A. Fornace A.J. Curr. Opin. Genet. Dev. 2002; 12: 92-97Crossref PubMed Scopus (155) Google Scholar, 17.Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar).These pathways all target the key G2/M transitional regulators, the cdc25 family of dual specificity phosphatases. The cdc25s activate the mitotic cdk complexes by dephosphorylating the inhibitory Thr14 and Tyr15 residues on cdk1 and cdk2. All three cdc25 isoforms appear to have roles in G2/M progression, although only depletion of cdc25A and cdc25B delays entry into mitosis (18.Lindqvist A. Källström H. Lundgren A. Barsoum E. Rosenthal C.K. J. Cell Biol. 2005; 171: 35-45Crossref PubMed Scopus (148) Google Scholar). Both cdc25A and cdc25B are unstable proteins, and their activity is in part regulated by their stability, which is increased in G2/M and decreased in response to stresses (19.Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar, 20.Xiao Z. Chen Z. Gunasekera A.H. Sowin T.J. Rosenberg S.H. Fesik S. Zhang H. J. Biol. Chem. 2003; 278: 21767-21773Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 21.Uto K. Inoue D. Shimuta K. Nakajo N. Sagata N. EMBO J. 2004; 23: 3386-3396Crossref PubMed Scopus (82) Google Scholar). All three cdc25 isoforms are targets for checkpoint kinase inactivation (22.Boutros R. Dozier C. Ducommun B. Curr. Opin. Cell Biol. 2006; 18: 185-191Crossref PubMed Scopus (342) Google Scholar). Cdc25A is destabilized by Chk1 phosphorylation in response to DNA damage (23.Mailand N. Falck J. Lukas C. Syljuâsen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (641) Google Scholar). Cdc25B is specifically required for exit from the G2 phase checkpoint arrest (24.van Vugt M.A. Brás A. Medema R.H. Mol. Cell. 2004; 15: 799-811Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), and its stability has also been linked to responses to damage (25.Bansal P. Lazo J.S. Cancer Res. 2007; 67: 3356-3363Crossref PubMed Scopus (34) Google Scholar). The possibility that MAPK signaling induces a G2 delay via the ATM/ATR, Chk1/2, or p38MAPK checkpoint pathways has not been reported. Here, we demonstrate that the MAPK signaling-induced G2 phase delay is independent of usual G2 checkpoint mechanisms, but instead is a consequence of MEK1-dependent destabilization of the critical G2/M regulator cdc25B.DISCUSSIONIn this report we have demonstrated that activation of MAPK pathway signaling induces a G2 phase delay via a MEK1-dependent mechanism culminating in the rapid destabilization of the critical G2/M regulator cdc25B. Depletion and inhibition of cdc25B have previously been demonstrated to delay entry into mitosis (18.Lindqvist A. Källström H. Lundgren A. Barsoum E. Rosenthal C.K. J. Cell Biol. 2005; 171: 35-45Crossref PubMed Scopus (148) Google Scholar, 39.Goldstone S. Pavey S. Forrest A. Sinnamon J. Gabrielli B. Oncogene. 2001; 20: 921-932Crossref PubMed Scopus (75) Google Scholar). Cdc25B cooperates with cdc25A in promoting normal G2/M progression and hence induces only relatively short delay. However, cdc25B is specifically required for exit from a G2 phase checkpoint arrest, and its depletion in this case causes a more significant delay in entry into mitosis (24.van Vugt M.A. Brás A. Medema R.H. Mol. Cell. 2004; 15: 799-811Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). The relatively transient delay induced with EGF or TPA in normal cycling cells and more robust delay observed in the G2 phase checkpoint-arrested cells driven into mitosis with caffeine exactly mirror the effects reported with siRNA-mediated depletion of cdc25B in these identical systems (18.Lindqvist A. Källström H. Lundgren A. Barsoum E. Rosenthal C.K. J. Cell Biol. 2005; 171: 35-45Crossref PubMed Scopus (148) Google Scholar, 24.van Vugt M.A. Brás A. Medema R.H. Mol. Cell. 2004; 15: 799-811Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). The lack of involvement of established pathways that promote a G2 phase delay, ATM/ATR, Chk1/2, and p38MAPK checkpoint signaling is not surprising because these pathways impose a G2 phase delay by regulating cdc25B activity (17.Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 21.Uto K. Inoue D. Shimuta K. Nakajo N. Sagata N. EMBO J. 2004; 23: 3386-3396Crossref PubMed Scopus (82) Google Scholar, 40.Schmitt E. Boutros R. Froment C. Monsarrat B. Ducommun B. Dozier C. J. Cell Sci. 2006; 119: 4269-4275Crossref PubMed Scopus (68) Google Scholar, 41.Boutros R. Lobjois V. Ducommun B. Nat. Rev. Cancer. 2007; 7: 495-507Crossref PubMed Scopus (545) Google Scholar).The contribution of upstream signaling through the MAPK pathway to the G2 phase delay is demonstrated by the ability of both TPA and EGF to impose the delay, and the relatively short lived effect EGF compared with TPA reflects the relative duration of MAPK signaling generated by each stimulus. The discovery that MEK1 but not MEK2 depletion blocked the EGF- and TPA-induced destabilization of cdc25B and G2 phase delay clearly demonstrates the involvement of MEK1 in this mechanism. In Xenopus extracts, MAPK activation causes a G2 phase delay, attributed to ERK phosphorylation of Wee1, and phosphorylation of cdc25C by p90RSK, a downstream effector of ERK activity (42.Walter S.A. Guadagno S.N. Ferrell Jr., J.E. Mol. Biol. Cell. 2000; 11: 887-896Crossref PubMed Scopus (51) Google Scholar, 43.Chun J. Chau A.S. Maingat F.G. Edmonds S.D. Ostergaard H.L. Shibuya E.K. Cell Cycle. 2005; 4: 148-154Crossref PubMed Scopus (19) Google Scholar). There is contradictory evidence of a role for MAPK signaling in G2 and mitosis in mammalian cells (5.Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (122) Google Scholar, 6.Wright 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 (157) Google Scholar, 8.Shinohara M. Mikhailov A.V. Aguirre-Ghiso J.A. Rieder C.L. Mol. Biol. Cell. 2006; 17: 5227-5240Crossref PubMed Scopus (28) Google Scholar), but recent evidence suggests that some of this maybe attributed to cell lineage-dependent differences (44.Dumesic P.A. Scholl F.A. Barragan D.I. Khavari P.A. J. Cell Biol. 2009; 185: 409-422Crossref PubMed Scopus (74) Google Scholar). MAPK signaling has also been implicated in the G2 arrest associated with overexpression of BRCA1 and p14ARF, although these mechanisms involved Chk1, differentiating them from the present study (45.Yan Y. Spieker R.S. Kim M. Stoeger S.M. Cowan K.H. Oncogene. 2005; 24: 3285-3296Crossref PubMed Scopus (50) Google Scholar, 46.Eymin B. Claverie P. Salon C. Brambilla C. Brambilla E. Gazzeri S. Cell Cycle. 2006; 5: 759-765Crossref PubMed Scopus (38) Google Scholar).The mechanism by which MEK1 destabilizes cdc25B appears to be through phosphorylation of Ser249. Cdc25B stability is regulated in part by the Skp1/Cullin1/F-box E3 ubiquitin ligase β-TrCP, which recognizes a DDG motif in the N-terminal half of the protein (38.Kanemori Y. Uto K. Sagata N. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6279-6284Crossref PubMed Scopus (97) Google Scholar). Interestingly, cdc25A, which is also regulated by β-TrCP, is the target for ERK-dependent phosphorylation, which accelerates its degradation in a manner similar to cdk1- and Chk1- dependent phosphorylation (47.Isoda M. Kanemori Y. Nakajo N. Uchida S. Yamashita K. Ueno H. Sagata N. Mol. Biol. Cell. 2009; 20: 2186-2195Crossref PubMed Scopus (33) Google Scholar). The sensitivity of the Ser249 site to the MEK inhibitor U0126 and the fact that it was originally identified as a p38MAPK-dependent site implicate ERK as being responsible for the Ser249 phosphorylation. p38MAPK and ERK have similar phosphorylation site determinants with their in vivo specificity determined by specific docking site interactions (48.Mayor Jr., F. Jurado-Pueyo M. Campos P.M. Murga C. Cell Cycle. 2007; 6: 528-533Crossref PubMed Scopus (37) Google Scholar). Additionally, TPA treatment rapidly inactivated p38MAPK, indicating that p38MAPK or MK2 was unlikely to be responsible for the phosphorylation observed (data not shown). Interestingly, Cdc25A destabilization in response to MAPK pathway activation was not detected in the current study. The reason for this is unclear. The inability of the MEK inhibitor U0126 to rescue the TPA/EGF-stimulated G2 phase delay and cdc25B destabilization completely may point to mechanisms in addition to decreased stability of cdc25B being involved in the delay. The ability of MEK1 depletion to block the loss of cdc25B completely indicates that the effect is MEK1-dependent, with the Ser249 phosphorylation and resultant destabilization likely to depend on ERK activation. However, cdc25B is an unstable protein with a half-life of less than 30 min in G2 phase cells (38.Kanemori Y. Uto K. Sagata N. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6279-6284Crossref PubMed Scopus (97) Google Scholar). It is possible that in addition to the ERK-dependent Ser249 phosphorylation and destabilization of cdc25B, MEK1 also blocks de novo synthesis of cdc25B. This would also effectively deplete cdc25B levels and could act in cooperation with the destabilization of the preexisting pool of cdc25B to maintain the G2 phase delay. The failure of the MEK inhibitor to rescue cdc25B levels completely suggests that the proposed block in de novo synthesis is independent of MEK1 catalytic activity.Although there appears to be significant functional redundancy between MEK1 and MEK2, a MEK1-specific function has recently been reported. Knock-out of MEK1, but not MEK2, reduced tumor formation in a chemically induced mouse skin cancer model (49.Scholl F.A. Dumesic P.A. Barragan D.I. Harada K. Charron J. Khavari P.A. Cancer Res. 2009; 69: 3772-3778Crossref PubMed Scopus (43) Google Scholar), suggesting a specific role for MEK1 in response to DNA-damaging agents. Our finding that the MEK1-dependent signaling, by targeting cdc25B stability, was capable of influencing the timing of exit from a G2 phase DNA damage checkpoint arrest may contribute directly to this MEK1-specific effect on tumor formation.The nature of the signal that stimulates the MEK1-dependent G2 delay mechanism we have defined is at present unknown. We have demonstrated that it responds to external signals such as growth factors and other factors that stimulate MAPK signaling. Recently, hepatocyte growth factor used at physiological levels was shown to produce a similar G2 phase delay (50.Nam H.J. Kim S. Lee M.W. Lee B.S. Hara T. Saya H. Cho H. Lee J.H. Cell. Signal. 2008; 20: 1349-1358Crossref PubMed Scopus (26) Google Scholar). Other stresses affecting the cell microenvironment or its interactions with neighboring cells that generate MAPK-dependent signaling (51.McKay M.M. Morrison D.K. Oncogene. 2007; 26: 3113-3121Crossref PubMed Scopus (446) Google Scholar) would influence checkpoint exit though this MEK1-dependent mechanism. Thus, in response to stresses that produce both intracellular damage that triggers a G2 phase checkpoint response through ATM/ATR signaling, and extracellular stress such as tissue damage triggering MAPK pathway activation, entry into mitosis would be delayed until both intracellular and extracellular stresses are resolved. This coordination between responses to intracellular and extracellular stress signals ensures that entry into mitosis only proceeds in optimal conditions to ensure the integrity of mitosis and proliferation of tissue following damage.In conclusion, we have demonstrated that activation of MAPK signaling in G2 phase cells induces a G2 phase delay through a MEK1-dependent pathway. This pathway destabilizes cdc25B protein by increased phosphorylation of Ser249, and it is the degradation of cdc25B that is responsible for the G2 phase delay observed in normally cycling cells, and the sensitivity of cells attempting to exit a G2 phase checkpoint arrest to MAPK signaling. Signaling through the MAPK pathway is critical for many biological activities, and upstream components of this pathway, notably Ras and Raf, are common targets for activating mutations in cancer. The demonstration that MEK1 can regulate normal G2/M cell cycle and perhaps, more importantly, checkpoint mechanisms, suggests a novel mechanism by which these mutations can promote neoplastic transformation. IntroductionThe canonical MAPK 5The abbreviations used are: MAPKmitogen-activated protein kinasecdkcyclin-dependent kinaseEGFepidermal growth factorERKextracellular signal-regulated kinaseGFPgreen fluorescent proteinMEFmouse embryonic fibroblastMEKMAPK/ERK kinasePKCprotein kinase CsiRNAsmall interfering RNATPA12-O-tetradecanoylphorbol-13-acetateATMataxia telangiectasia mutatedATRATM-Rad3 related. pathway of Ras, Raf, MEK, and ERK provides a sensitive mechanism for transducing extracellular signals critical for cell growth and development (1.Harding A. Tian T. Westbury E. Frische E. Hancock J.F. Curr. Biol. 2005; 15: 869-873Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). ERK-dependent phosphorylation of a broad range of substrates is the primary signaling output of the pathway (2.Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2248) Google Scholar). The ability of the MAPK pathway to influence entry into the cell cycle has been well established (3.Edelmann H.M. Kühne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4.Lavoie J.N. L'Allemain G. Brunet A. Müller R. Pouysségur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 5.Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (122) Google Scholar). Signaling through this pathway also has a role in G2 phase (6.Wright 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 (157) Google Scholar, 7.Liu X. Yan S. Zhou T. Terada Y. Erikson R.L. Oncogene. 2004; 23: 763-776Crossref PubMed Scopus (132) Google Scholar, 8.Shinohara M. Mikhailov A.V. Aguirre-Ghiso J.A. Rieder C.L. Mol. Biol. Cell. 2006; 17: 5227-5240Crossref PubMed Scopus (28) Google Scholar), possibly to regulate Golgi disassembly (9.Colanzi A. Sutterlin C. Malhotra V. J. Cell Biol. 2003; 161: 27-32Crossref PubMed Scopus (58) Google Scholar).Growth factors activate the canonical MAPK pathway through specific receptors, whereas the tumor-promoting phorbol esters activate this pathway via PKC (10.El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). When either growth factors or phorbol esters are added in G2 phase they can induce a G2 phase cell cycle delay (11.Barth H. Kinzel V. Exp. Cell Res. 1994; 212: 383-388Crossref PubMed Scopus (23) Google Scholar, 12.Barth H. Kinzel V. J. Cell. Physiol. 1995; 162: 44-51Crossref PubMed Scopus (10) Google Scholar); however, the exact mechanism of the delay is poorly understood. Activation of the MAPK pathway by growth factors and phorbol esters has been implicated in the G2 phase delay (13.Dangi S. Chen F.M. Shapiro P. Cell Prolif. 2006; 39: 261-279Crossref PubMed Scopus (33) Google Scholar), with the ERK-dependent up-regulation of the cdk inhibitor p21WAF1 identified as a key component of the delay (13.Dangi S. Chen F.M. Shapiro P. Cell Prolif. 2006; 39: 261-279Crossref PubMed Scopus (33) Google Scholar). Other mechanisms, including increased protein phosphatase 2A activity, have also been proposed (14.Klingler-Hoffmann M. Barth H. Richards J. König N. Kinzel V. Eur. J. Cell Biol. 2005; 84: 719-732Crossref PubMed Scopus (8) Google Scholar).Cells respond to a variety of stresses by imposing a G2 phase delay through the action of cell cycle checkpoint mechanisms. The checkpoint mechanisms impose the arrest by blocking activation of cyclin B/cdk1, the driver of mitosis. Major checkpoint mechanisms demonstrated to impose a G2 phase arrest in response to DNA damage are the ATM and related ATR-dependent pathways. ATM and ATR are the apical components of pathways that signal through Chk1 and Chk2 to block cdc25-dependent activation of the mitotic cyclin/cdks (15.O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). The p38MAPK-MAPKAPK2 pathway is involved in a separate G2 arrest pathway (16.Bulavin D.V. Amundson S.A. Fornace A.J. Curr. Opin. Genet. Dev. 2002; 12: 92-97Crossref PubMed Scopus (155) Google Scholar, 17.Manke I.A. Nguyen A. Lim D. Stewart M.Q. Elia A.E. Yaffe M.B. Mol. Cell. 2005; 17: 37-48Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar).These pathways all target the key G2/M transitional regulators, the cdc25 family of dual specificity phosphatases. The cdc25s activate the mitotic cdk complexes by dephosphorylating the inhibitory Thr14 and Tyr15 residues on cdk1 and cdk2. All three cdc25 isoforms appear to have roles in G2/M progression, although only depletion of cdc25A and cdc25B delays entry into mitosis (18.Lindqvist A. Källström H. Lundgren A. Barsoum E. Rosenthal C.K. J. Cell Biol. 2005; 171: 35-45Crossref PubMed Scopus (148) Google Scholar). Both cdc25A and cdc25B are unstable proteins, and their activity is in part regulated by their stability, which is increased in G2/M and decreased in response to stresses (19.Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar, 20.Xiao Z. Chen Z. Gunasekera A.H. Sowin T.J. Rosenberg S.H. Fesik S. Zhang H. J. Biol. Chem. 2003; 278: 21767-21773Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 21.Uto K. Inoue D. Shimuta K. Nakajo N. Sagata N. EMBO J. 2004; 23: 3386-3396Crossref PubMed Scopus (82) Google Scholar). All three cdc25 isoforms are targets for checkpoint kinase inactivation (22.Boutros R. Dozier C. Ducommun B. Curr. Opin. Cell Biol. 2006; 18: 185-191Crossref PubMed Scopus (342) Google Scholar). Cdc25A is destabilized by Chk1 phosphorylation in response to DNA damage (23.Mailand N. Falck J. Lukas C. Syljuâsen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (641) Google Scholar). Cdc25B is specifically required for exit from the G2 phase checkpoint arrest (24.van Vugt M.A. Brás A. Medema R.H. Mol. Cell. 2004; 15: 799-811Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), and its stability has also been linked to responses to damage (25.Bansal P. Lazo J.S. Cancer Res. 2007; 67: 3356-3363Crossref PubMed Scopus (34) Google Scholar). The possibility that MAPK signaling induces a G2 delay via the ATM/ATR, Chk1/2, or p38MAPK checkpoint pathways has not been reported. Here, we demonstrate that the MAPK signaling-induced G2 phase delay is independent of usual G2 checkpoint mechanisms, but instead is a consequence of MEK1-dependent destabilization of the critical G2/M regulator cdc25B. The canonical MAPK 5The abbreviations used are: MAPKmitogen-activated protein kinasecdkcyclin-dependent kinaseEGFepidermal growth factorERKextracellular signal-regulated kinaseGFPgreen fluorescent proteinMEFmouse embryonic fibroblastMEKMAPK/ERK kinasePKCprotein kinase CsiRNAsmall interfering RNATPA12-O-tetradecanoylphorbol-13-acetateATMataxia telangiectasia mutatedATRATM-Rad3 related. pathway of Ras, Raf, MEK, and ERK provides a sensitive mechanism for transducing extracellular signals critical for cell growth and development (1.Harding A. Tian T. Westbury E. Frische E. Hancock J.F. Curr. Biol. 2005; 15: 869-873Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). ERK-dependent phosphorylation of a broad range of substrates is the primary signaling output of the pathway (2.Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2248) Google Scholar). The ability of the MAPK pathway to influence entry into the cell cycle has been well established (3.Edelmann H.M. Kühne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4.Lavoie J.N. L'Allemain G. Brunet A. Müller R. Pouysségur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 5.Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (122) Google Scholar). Signaling through this pathway also has a role in G2 phase (6.Wright 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 (157) Google Scholar, 7.Liu X. Yan S. Zhou T. Terada Y. Erikson R.L. Oncogene. 2004; 23: 763-776Crossref PubMed Scopus (132) Google Scholar, 8.Shinohara M. Mikhailov A.V. Aguirre-Ghiso J.A. Rieder C.L. Mol. Biol. Cell. 2006; 17: 5227-5240Crossref PubMed Scopus (28) Google Scholar), possibly to regulate Golgi disassembly (9.Colanzi A. Sutterlin C. Malhotra V. J. Cell Biol. 2003; 161: 27-32Crossref PubMed Scopus (58) Google Scholar). mitogen-activated protein kinase cyclin-dependent kinase epidermal growth factor extracellular signal-regulated kinase green fluorescent protein mouse embryonic fibroblast MAPK/ERK kinase protein kinase C small interfering RNA 12-O-tetradecanoylphorbol-13-acetate ataxia telangiectasia mutated ATM-Rad3 related. Growth factors activate the canonical MAPK pathway through specific receptors, whereas the tumor-promoting phorbol esters activate this pathway via PKC (10.El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). When either growth factors or phorbol esters are added in G2 phase they can induce a G2 phase cell cycle delay (11.Barth H. Kinzel V. Exp. Cell Res. 1994; 212: 383-388Crossref PubMed Scopus (23) Google Scholar, 12.Barth H. Kinzel V. J. Cell. Physiol. 1995; 162: 44-51Crossref PubMed Scopus (10) Google Scholar); however, the exact mechanism of the delay is poorly understood. Activation of the MAPK pathway by growth factors and phorbol esters has been implicated in the G2 phase delay (13.Dangi S. Chen F.M. Shapiro P. 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