The Xenopus Suc1/Cks Protein Promotes the Phosphorylation of G2/M Regulators

Abstract

The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation. These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2. The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation. These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2. M-phase promoting factor The entry into mitosis in eukaryotic cells is controlled by maturation or M-phase promoting factor (MPF)1 (1Morgan D.O. Annu. Rev. Cell. Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1802) Google Scholar). InXenopus egg extracts, MPF is a trimeric complex consisting of the protein kinase Cdc2, a B-type cyclin, and a small 9-kDa subunit called Xe-p9 (p9), a homolog of the Suc1/Cks protein (2Patra D. Dunphy W.G. Genes Dev. 1996; 10: 1503-1515Crossref PubMed Scopus (81) Google Scholar). MPF acts by carrying out key regulatory phosphorylations on various substrates that are involved in mitotic processes such as nuclear disassembly and chromosome transmission. In addition, MPF participates in regulatory circuits that control its “autocatalytic” activation at the G2/M transition (3Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (564) Google Scholar, 4Izumi T. Maller J.L. Mol. Biol. Cell. 1993; 4: 1337-1350Crossref PubMed Scopus (253) Google Scholar) and its inactivation at the conclusion of M-phase, respectively (1Morgan D.O. Annu. Rev. Cell. Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1802) Google Scholar). During interphase, Cdc2 is kept inactive because of inhibitory phosphorylations on its Tyr-15 and Thr-14 residues. These modifications are catalyzed by the kinases Wee1 and Myt1 (1Morgan D.O. Annu. Rev. Cell. Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1802) Google Scholar). The dual-specificity phosphatase Cdc25 removes both inhibitory phosphates when the conditions are appropriate for mitosis, thereby activating Cdc2 (1Morgan D.O. Annu. Rev. Cell. Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1802) Google Scholar). Cdc25, Myt1, and Wee1 are all highly regulated during the cell cycle (5Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (336) Google Scholar, 6Izumi T. Walker D.H. Maller J.L. Mol. Biol. Cell. 1992; 3: 927-939Crossref PubMed Scopus (233) Google Scholar, 7Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (269) Google Scholar, 8Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (362) Google Scholar, 9McGowan C.H. Russell P. EMBO J. 1995; 14: 2166-2175Crossref PubMed Scopus (243) Google Scholar, 10Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (538) Google Scholar). For example, these enzymes become highly phosphorylated at mitosis and concomitantly undergo large changes in their catalytic activities. In particular, the kinases Cdc2/cyclin B and Plx1 collaborate to carry out the stimulatory phosphorylations of Cdc25 (3Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (564) Google Scholar, 4Izumi T. Maller J.L. Mol. Biol. Cell. 1993; 4: 1337-1350Crossref PubMed Scopus (253) Google Scholar, 5Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (336) Google Scholar, 11Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (469) Google Scholar). Conversely, the Cdc2-specific kinase activities of both Myt1 and Wee1 are strongly down-regulated by phosphorylation at mitosis (7Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (269) Google Scholar, 8Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (362) Google Scholar, 9McGowan C.H. Russell P. EMBO J. 1995; 14: 2166-2175Crossref PubMed Scopus (243) Google Scholar, 10Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (538) Google Scholar). The Suc1/Cks protein is also essential for the proper regulation of Cdc2 in various species (2Patra D. Dunphy W.G. Genes Dev. 1996; 10: 1503-1515Crossref PubMed Scopus (81) Google Scholar, 12Moreno S. Hayles J. Nurse P. Cell. 1989; 58: 361-372Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 13Tang Y. Reed S.I. Genes Dev. 1993; 7: 822-832Crossref PubMed Scopus (83) Google Scholar, 14Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15Kaiser P. Moncollin V. Clarke D.J. Watson M.H. Bertolaet B.L. Reed S.I. Bailly E. Genes Dev. 1999; 13: 1190-1202Crossref PubMed Scopus (76) Google Scholar). In Xenopus egg extracts, p9 is required for both entry into and exit from mitosis (2Patra D. Dunphy W.G. Genes Dev. 1996; 10: 1503-1515Crossref PubMed Scopus (81) Google Scholar). The role of p9 in mitotic exit is now understood in some detail. In particular, p9 is necessary for the destruction of cyclin B and the phosphorylation of various proteins associated with ubiquitin-mediated proteolysis (e.g. Cdc27 and BIME) (16Patra D. Dunphy W.G. Genes Dev. 1998; 12: 2549-2559Crossref PubMed Scopus (122) Google Scholar). In the case of Cdc27, p9 was shown to strongly enhance the ability of Cdc2/cyclin B to phosphorylate recombinant Cdc27 without affecting the intrinsic catalytic activity of Cdc2, indicating that p9 facilitates substrate recognition (16Patra D. Dunphy W.G. Genes Dev. 1998; 12: 2549-2559Crossref PubMed Scopus (122) Google Scholar, 17Shteinberg M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 257: 12-18Crossref PubMed Scopus (36) Google Scholar). Significantly, immunodepletion of p9 also prevents the entry of Xenopus interphase egg extracts into M-phase, but the molecular basis of this defect has not been resolved. In the absence of p9, the Cdc2/cyclin B complex accumulates in its inactive form that is phosphorylated on Tyr-15 and Thr-14, suggesting that p9 in some manner regulates the dephosphorylation of these residues. The Pin1 protein has recently emerged as another potential regulator of mitotic progression (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar, 19Ranganathan R. Lu K.P. Hunter T. Noel J.P. Cell. 1997; 89: 875-886Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar, 21Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar, 22Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (592) Google Scholar). The human Pin1 protein was identified in a yeast two-hybrid assay by virtue of its ability to interact with the mitotic regulatory kinase NIMA (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar). Pin1 is a nuclear protein that has two structural domains: an N-terminal WW domain that is involved in protein-protein interactions and a C-terminal prolyl-isomerase domain. Depletion of Pin1 from HeLa cells results in a mitotic arrest, whereas overexpression of Pin1 in HeLa cells causes a G2 arrest (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar). Similarly, the addition of excess recombinant human His6-Pin1 protein to cycling Xenopus egg extracts can block the entry of these extracts into mitosis (20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar, 21Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar). These studies indicated that Pin1 can impinge upon the Cdc2/cyclin B-dependent pathway of mitotic regulation. In this report, we have examined the requirement for p9 in regulating the G2/M transition. We find that p9 significantly stimulates the ability of a Cdc2/cyclin B complex to phosphorylate the mitotic regulators Cdc25, Myt1, and Wee1. This observation suggests that p9 plays an indispensable role in the “autocatalytic” activation of MPF at the G2/M transition. In the course of these studies, we also observed that excess Pin1 protein can antagonize the stimulatory effect of p9 on the phosphorylation of the regulators of Cdc2, suggesting that Pin1 may likewise participate in the activation of Cdc2. His6-Cdc25 and His6-Myt1 proteins were purified from baculovirus-infected Sf9 cells as described (10Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (538) Google Scholar, 11Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (469) Google Scholar). Phosphorylation of these proteins was analyzed using 8% SDS gel electrophoresis. 35S-Labeled Xenopus Wee1 was synthesized using pET3a-XeWee1 as the template (7Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (269) Google Scholar) and the TNT in vitro transcription/translation system (Promega Corp.) in the presence of [35S]Translabel (ICN Biomedicals, Inc., Irvine, CA). Analysis of the phosphorylation products was performed by SDS-gel electrophoresis with a 10% polyacrylamide gel containing 0.357% piperazine diacrylamide (Bio-Rad) instead of 0.267% bisacrylamide. Wild-type His6-Pin1 was expressed and purified from Escherichia coliBL21(DE3)pLysS cells using the pET28a-His6-Pin1 construct (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar) as described (19Ranganathan R. Lu K.P. Hunter T. Noel J.P. Cell. 1997; 89: 875-886Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar) except that the protein was induced at an optical density of 0.5 (600 nm). A plasmid encoding the mutant His6-Pin1-W34A protein was constructed with the QuikChangeTM Site-directed Mutagenesis kit (Stratagene), using pET28a-His6-Pin1 as the template (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar) and two oligonucleotides with the following sequences: 5′-CACATCACTAACGCTAGCCAGGCGGAGCGGCCCAGCG-3′ and 5′-CGCTGGGCCGCTCCGCCTGGCTAGCGTTAGTGATGTG-3′. This procedure resulted in the conversion of the Trp-34 to Ala (100TGG103 to100GCG103) as well as the insertion of a silent mutation in the coding region of His6-Pin1 (91GCC93 to 91GCT93) that allowed for the identification of the mutant clone by restriction analysis with NheI and EcoRV. These mutations were further confirmed by sequencing. The mutant His6-Pin1-W34A protein was then expressed and purified as described above for the wild-type protein. To assess the role that p9 plays in mitotic entry, we examined whether p9 would affect the Cdc2-dependent phosphorylation of various regulators of the G2/M transition. For this purpose, we incubated recombinant Xenopus Cdc25, Myt1, and Wee1 with a dimeric Cdc2/Δcyclin B complex or trimeric p9/Cdc2/Δcyclin B complex. For Cdc25 and Myt1, phosphorylation was assessed by monitoring 32P incorporation and mobility during gel electrophoresis. As shown in Fig. 1 (top and middle panels), p9 strongly stimulated the incorporation of radioactive phosphate into both His6-Cdc25 and His6-Myt1. Furthermore, treatment with p9/Cdc2/Δcyclin B, but not Cdc2/Δcyclin B, resulted in a large decrease in the electrophoretic mobilities of both Cdc25 and Myt1, which is characteristic of the mitotic forms of these proteins. The presence of p9 in the Cdc2/Δcyclin B complex consistently resulted in a 4- to 5-fold increase in the appearance of the hyperphosphorylated forms of Cdc25 and Myt1. Because Xenopus Wee1 possesses substantial autophosphorylation activity and undergoes less extensive modification at mitosis, we assessed its phosphorylation by monitoring the shifting of 35S-Wee1 in SDS gels under modified electrophoretic conditions (see “Experimental Procedures”). We observed that p9 also significantly increased the phosphorylation of35S-Wee1 by Cdc2/Δcyclin B, but to a lesser degree than for Cdc25 and Myt1 (Fig. 1, bottom panel). The prolyl isomerase Pin1 binds to the phosphorylated forms of Cdc25, Myt1, and Wee1, as well as other mitotic phosphoproteins near the beginning of M-phase (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar, 19Ranganathan R. Lu K.P. Hunter T. Noel J.P. Cell. 1997; 89: 875-886Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar, 21Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar, 22Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (592) Google Scholar). Overexpression of Pin1 in human cells (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar) and Xenopus egg extracts inhibits the entry into mitosis (20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar, 21Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar). Depletion of Pin1 from human cells by expression of an antisense construct and deletion of the gene encoding the budding yeast Pin1 homologue Ess1 both result in a defect in the progression through mitosis (18Lu K.P. Hanes S.D. Hunter T. Science. 1996; 380: 544-547Google Scholar). Although the precise physiological role of Pin1 has not been established, these data collectively suggest that Pin1, like p9, plays some role both in the entry into and progression through mitosis. We asked whether Pin1 might affect the ability of p9 to stimulate substrate recognition by Cdc2/Δcyclin B. Recombinant His6-Pin1 (100 μg/ml) completely abolished the stimulatory effect of p9 on the Cdc2-catalyzed phosphorylation of His6-Cdc25 and His6-Myt1 (Fig. 2 A). In dose-response studies with His6-Cdc25 as the substrate, we observed that half-maximal inhibition of the p9-stimulated phosphorylation of Cdc25 occurred at ∼50 μg/ml His6-Pin1 (Fig. 2 B). Previously, the endogenous concentration of Pin1 in Xenopus egg extracts was estimated to be 0.5 μm or 9 μg/ml (20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar). Furthermore, recombinant Pin1 at a final concentration of 10 μm (180 μg/ml) completely blocked the entry of egg extracts into mitosis (20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar). Thus, the concentration of His6-Pin1 that inhibits the p9-dependent phosphorylation of Cdc25 by Cdc2/Δcyclin B is within the range previously established to block mitosis in egg extracts. In control experiments, we examined whether His6-Pin1 would affect the catalytic activity of p9/Cdc2/Δcyclin B toward the generic substrate histone H1. As shown in Fig. 2 C, up to 100 μg/ml His6-Pin1 had no effect on the ability of p9/Cdc2/Δcyclin B to phosphorylate histone H1. As reported previously (16Patra D. Dunphy W.G. Genes Dev. 1998; 12: 2549-2559Crossref PubMed Scopus (122) Google Scholar), Cdc2/Δcyclin B and p9/Cdc2/Δcyclin B phosphorylated histone H1 with equal efficiency (Fig. 2 C, compare lanes a and b). The Cdc25 protein can also be phosphorylated and regulated by the Polo-like kinases (Plx1 in Xenopus) (11Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (469) Google Scholar). However, His6-Pin1 (100 μg/ml) had no inhibitory effect on the ability of Plx1 to phosphorylate Cdc25 in vitro (data not shown). Finally, we examined the possibility that His6-Pin1 might disrupt the binding of p9 to Cdc2/Δcyclin B because p9 does possess a proline-containing region that has been implicated in controlling the binding of Suc1/Cks proteins to cyclin-dependent kinases (23Bourne Y. Arvai A.S. Bernstein S.L. Watson M.H. Reed S.I. Endicott J.E. Noble M.E. Johnson L.N. Tainer J.A. Proc. Natl. Acad. Sci. 1995; 92: 10232-10236Crossref PubMed Scopus (67) Google Scholar, 24Endicott J.A. Noble M.E. Garman E.F. Brown N. Rasmussen B. Nurse P. Johnson L.N. EMBO J. 1995; 14: 1004-1014Crossref PubMed Scopus (67) Google Scholar, 25Bourne Y. Watson M.H. Hickey M.J. Holmes W. Rocque W. Reed S.I. Tainer J.A. Cell. 1996; 84: 863-874Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). By immunoblotting with anti-p9 antibodies (2Patra D. Dunphy W.G. Genes Dev. 1996; 10: 1503-1515Crossref PubMed Scopus (81) Google Scholar), we observed that His6-Pin1 (100 μg/ml) did not affect the binding of p9 to Cdc2/Δcyclin B (data not shown). Pin1 consists of a carboxyl-terminal prolyl isomerase domain and an amino-terminal WW domain that binds to peptides containing phosphoserine or phosphothreonine (22Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (592) Google Scholar). Its WW domain is critical for the association of Pin1 with mitotic phosphoproteins such as Cdc25. To ask whether the WW domain of Pin1 is necessary for its ability to antagonize p9, we prepared the mutant His6-Pin1-W34A in which the function of the WW domain was abolished by mutagenesis of Trp-34 to Ala. As shown in Fig. 3, the mutant His6-Pin1-W34A, in contrast to wild-type His6-Pin1, was not able to inhibit the p9-stimulated hyperphosphorylation of Cdc25. In contrast, a form of Pin1 with a mutation in the prolyl isomerase domain (Pin1-H59A) (19Ranganathan R. Lu K.P. Hunter T. Noel J.P. Cell. 1997; 89: 875-886Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar) could still inhibit the stimulatory effect of p9 on the phosphorylation of Cdc25 (data not shown). Thus, the WW domain of Pin1 is crucial for its ability to impair the phosphorylation of Cdc25. In summary, one finding of this report is that p9 strongly enhances the ability of Cdc2 to phosphorylate its key upstream regulators (e.g. Cdc25, Myt1, and Wee1) (Fig. 4). Mitotic hyperphosphorylation results in up-regulation of Cdc25 (3Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (564) Google Scholar, 4Izumi T. Maller J.L. Mol. Biol. Cell. 1993; 4: 1337-1350Crossref PubMed Scopus (253) Google Scholar, 5Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (336) Google Scholar, 6Izumi T. Walker D.H. Maller J.L. Mol. Biol. Cell. 1992; 3: 927-939Crossref PubMed Scopus (233) Google Scholar) and down-regulation of Wee1 (7Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (269) Google Scholar, 8Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (362) Google Scholar, 9McGowan C.H. Russell P. EMBO J. 1995; 14: 2166-2175Crossref PubMed Scopus (243) Google Scholar) and Myt1 (10Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (538) Google Scholar), respectively. Cdc2/cyclin B appears to play a crucial role in these mitotic hyperphosphorylations. Although Plx1 and possibly other kinases participate in this process, it is not fully clear whether Plx1 acts upstream or downstream of Cdc2. Taken together, our results strongly suggest that the arrest of p9-depletedXenopus egg extracts in interphase can be attributed at least in to a in the Cdc2-dependent hyperphosphorylation of Cdc25, Myt1, and Wee1. Cdc25 could not be activated and both Myt1 and Wee1 could not be inactivated in the absence of Thus, p9 be as an essential of p9 could play an in regulating the G2/M such as affecting the ability of Cdc25, Myt1, and Wee1 to Cdc2, but we have not been able to effect of p9 on the catalytic of Cdc25, Myt1, and Wee1 (data not shown). In structural studies, the Suc1/Cks protein has been shown to a potential Y. Watson M.H. Hickey M.J. Holmes W. Rocque W. Reed S.I. Tainer J.A. Cell. 1996; 84: 863-874Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). In this could p9 to phosphoproteins such as Cdc25, Myt1, and Wee1. In this p9 could stimulate the further phosphorylation of these proteins by Cdc2/cyclin B through its ability to increase the concentration of Cdc2 in to its The of p9 as a factor could to explain the of mitotic entry in Xenopus egg extracts. In this the activation of Cdc2 by cyclin B a critical concentration of cyclin B and a substantial the binding of cyclin B to Cdc2 and the activation of this complex M.J. M. M. Kirschner M.W. Cell. Full Text PDF PubMed Scopus Google Scholar). conclusion of this is that Pin1 can disrupt the stimulatory of p9 through the ability of its WW domain to to the regulators (e.g. Cdc25, Myt1, and Wee1) that are also substrates of B (Fig. 4). The ability of Pin1 to block the activation of Cdc25 and inactivation of Wee1 and Myt1 could explain why overexpression of Pin1 the entry into mitosis egg extracts and human are whether Pin1 can inhibit the phosphatase activity of Cdc25 (20Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (305) Google Scholar, 21Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar), but we have not been able to an inhibitory effect of human Pin1 on the catalytic activity Cdc25 (data not shown). Thus, Pin1 may be able to inhibit the catalytic function of Cdc25 under assay we suggest that overexpression of Pin1 mitotic entry by phosphorylation of the upstream regulators of Cdc2. These findings the possibility that p9 and Pin1 may play in controlling the G2/M transition. However, at this it is not known whether inhibition of mitotic entry by Pin1 could a physiological function of Pin1 or is instead a of its studies be required to the physiological role of Pin1 in cell cycle We Hunter for the plasmid encoding wild-type human His6-Pin1 and Lu for the mutant protein. We are to the of the Dunphy for on the