Greatwall dephosphorylation and inactivation upon mitotic exit is triggered by PP1
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Entry into mitosis is induced by the activation of cyclin-B-Cdk1 and Greatwall (Gwl; also known as MASTL in mammals) kinases. Cyclin-B-Cdk1 phosphorylates mitotic substrates, whereas Gwl activation promotes the phosphorylation of the small proteins Arpp19 and ENSA. Phosphorylated Arpp19 and/or ENSA bind to and inhibit PP2A comprising the B55 subunit (PP2A-B55; B55 is also known as PPP2R2A), the phosphatase responsible for cyclin-B-Cdk1 substrate dephosphorylation, allowing the stable phosphorylation of mitotic proteins. Upon mitotic exit, cyclin-B-Cdk1 and Gwl kinases are inactivated, and mitotic substrates are dephosphorylated. Here, we have identified protein phosphatase-1 (PP1) as the phosphatase involved in the dephosphorylation of the activating site (Ser875) of Gwl. Depletion of PP1 from meioticXenopusegg extracts maintains phosphorylation of Ser875, as well as the full activity of this kinase, resulting in a block of meiotic and mitotic exit. By contrast, preventing the reactivation of PP2A-B55 through the addition of a hyperactive Gwl mutant (GwlK72M) mainly affected Gwl dephosphorylation on Thr194, resulting in partial inactivation of Gwl and in the incomplete exit from mitosis or meiosis. We also show that when PP2A-B55 is fully reactivated by depleting Arpp19, this protein phosphatase is able to dephosphorylate both activating sites, even in the absence of PP1.Keywords:
Dephosphorylation
Mitotic exit
Cyclin B
Meiosis II
Cell growth prior to cell division is restricted by the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complexes. Recently, we identified that the death-effector domain (DED) containing protein, DEDD, acts as a novel inhibitor of mitotic Cdk1/cyclin B1, influencing cell size. Like cyclin B1, DEDD protein levels specifically peak during the G(2)/M phase. In the nucleus, DEDD associates with Cdk1/cyclin B1 complexes, via direct binding to cyclin B1, and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD(-/-)) embryonic fibroblasts is increased compared to that in DEDD(+/+) cells. This accelerates mitotic progression in DEDD(-/-) cells, with a shortened G(2)/M phase, reduced rRNA, and diminished cell volume. Likewise, DEDD(-/-) mice show decreased body and organ weights relative to DEDD(+/+) mice. Interestingly, the DED domain is not involved in the association of DEDD with Cdk1/cyclin B1, but is indispensable for the cell sizing function of DEDD. Together, in addition to the well-established machinery for activation of Cdk1 through dephosphorylation of its inhibitory-residues, we propose a novel mechanism for impeditive regulation of mitotic Cdk1/cyclin B1 mediated by DEDD within the nucleus, which allows sufficient cell growth prior to cell division.
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Cyclin B1
Cyclin B
Meiosis II
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Abstract Cyclin-dependent-kinases (CDKs) are essential for cell cycle progression. While dependence of CDK activity on Cyclin levels is established, molecular mechanisms that regulate their binding are less studied. Here, we show that CDKl:Cyclin-B interactions are regulated by acetylation, which was hitherto unknown. We demonstrate that cell cycle dependent acetylation of the evolutionarily conserved catalytic lysine in CDK1 or eliminating its charge state abrogates Cyclin-B binding. Opposing activities of SIRT1 and P300 regulate acetylation, which marks a reserved pool of CDK1. Our high resolution structural analyses into the formation of kinase competent CDK1: Cyclin-B complex have unveiled long-range effects of catalytic lysine in configuring the CDK1 interface for Cyclin-B binding. Cells expressing acetylation mimic mutant of Cdc2 in yeast are arrested in G2 and fail to divide. Thus, by illustrating cell cycle dependent deacetylation as a determinant of CDK1:Cyclin-B interaction, our results redefine the current model of CDK1 activation and cell cycle progression.
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Inactivation of cyclin-dependent kinase (Cdk) 1 promotes exit from mitosis and establishes G1. Proteolysis of cyclin B is the major known mechanism that turns off Cdk1 during mitotic exit. Here, we show that mitotic exit also activates pathways that catalyze inhibitory phosphorylation of Cdk1, a mechanism previously known to repress Cdk1 only during S and G2 phases of the cell cycle. We present evidence that down-regulation of Cdk1 activates Wee1 and Myt1 kinases and inhibits Cdc25 phosphatase during the M to G1 transition. If cyclin B/Cdk1 complex is present in G1, the inhibitory sites on Cdk1 become phosphorylated. Exit from mitosis induced by chemical Cdk inhibition can be reversed if cyclin B is preserved. However, this reversibility decreases with time after mitotic exit despite the continued presence of the cyclin. We show that this G1 block is due to phosphorylation of Cdk1 on inhibitory residues T14 and Y15. Chemical inhibition of Wee1 and Myt1 or expression of Cdk1 phosphorylation site mutants allows reversal to M phase even from late G1. This late Cdk1 reactivation often results in caspase-dependent cell death. Thus, in G1, the Cdk inhibitory phosphorylation pathway is functional and can lock Cdk1 in the inactive state.
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Mitotic exit
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// Fei-Ran Gong 1, 2, 3, 4, 5, * , Meng-Yao Wu 1, * , Meng Shen 1, * , Qiaoming Zhi 6 , Ze-Kuan Xu 7 , Rong Wang 1 , Wen-Jie Wang 1 , Yang Zong 7, 8 , Zeng-Liang Li 7 , Yadi Wu 9, 11 , Binhua P. Zhou 10, 11 , Kai Chen 1 , Min Tao 1, 12, 13, 14 , Wei Li 1, 14 1 Departments of Oncology, the First Affiliated Hospital of Soochow University, Suzhou, China 2 Departments of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China 3 Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China 4 Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, the First Affiliated Hospital of Soochow University, Suzhou, China 5 Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China 6 Department of General Surgery, the First Affiliated Hospital of Soochow University, Suzhou, China 7 Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China 8 Department of General Surgery, the Changshu No.1 People's Hospital, Changshu, China 9 Departments of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY, USA 10 Departments of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA 11 Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA 12 Jiangsu Institute of Clinical Immunology, Suzhou, China 13 Institute of Medical Biotechnology, Soochow University, Suzhou, China 14 PREMED Key Laboratory for Precision Medicine, Soochow University, Suzhou, China * These authors have contributed equally to this work Correspondence to: Wei Li, e-mail: liwei10@suda.edu.cn Min Tao, e-mail: mtao@medmail.com.cn ; e-mail: taomin@suda.edu.cn Keywords: PP2A, G2/M cell cycle arrest, JNK, CDK1, Sp1 Received: January 14, 2015 Accepted: May 14, 2015 Published: May 25, 2015 ABSTRACT Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.
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Protein phosphatase 1
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The molecular machinery controlling the meiotic, as well as mitotic, cell cycle is centered around the regulation of the activity of MPF, a complex composed of catalytic Cdc2 and the cyclin B regulatory subunit which triggers germinal vesicle breakdown and reentry into the meiotic cell cycle. MPF activity depends on the balance between Wee Kinase activity and cdc25B activity. Wee kinase phosphorylates and inactivates Cdc2. Conversely, Cdc25B dephosphorylates and activates cdc2. Although high levels of cAMP and protein kinase A (PKA) play a critical role in maintaining inactive Cdc2, the steps downstream of PKA in the oocyte remain unknown. In cultured PDE3A knock out (KO) oocytes (arrested at G2 stage), activities of MPF, Wee kinase and Cdc25B, and amounts of cyclin B1, Cdc2 and phospho-Cdc2(Thr14/Tyr15) were assessed. Consistent with our earlier data, during incubation of oocytes MPF activity increased in wild type (WT), but not in KO, oocytes. During a 4 hr incubation, Wee kinase activity decreased both in WT and KO oocytes, and Cdc25B activity increased in WT, but not in KO, oocytes. Whereas, during this time period, immunoreactive cyclin B1 increased in WT oocytes, in KO oocytes, it increased for 2 hours, and then significantly decreased. Phospho-Cdc2(Thr14/Tyr15) was decreased in WT, but not in KO oocytes. Our data indicate that Cdc25B, not Wee kinase, maybe a critical PKA substrate that directly or indirectly blocks MPF activity, and thereby induces arrest of oocytes at G2 stage. Since MPF, which could affect Cyclin B degradation via APC activation, is not activated in KO oocytes, other mechanisms for regulation of cyclin B degradation will be assessed.
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Cyclin B
Germinal vesicle
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SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition
The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1, and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 is non-essential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. After hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gβγ-PI3K pathway and an unidentified pathway downstream of Gβγ, called the atypical Gβγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.
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Cyclin B1
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