Plk1 Regulates Activation of the Anaphase Promoting Complex by Phosphorylating and Triggering SCF TrCP-dependent Destruction of the APC Inhibitor Emi1

Stanford University, Stanford, California, United States
Molecular Biology of the Cell (Impact Factor: 4.47). 01/2005; 15(12):5623-34. DOI: 10.1091/mbc.E04-07-0598
Source: PubMed


Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCF(betaTrCP) ubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, betaTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.

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Available from: David V Hansen, May 08, 2014
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    • "Both classic E3 ubiquitin ligases – SCF and APC complex – are closely associated with regulation of oocyte meiotic maturation, and Emi1 is a link protein between the two. The SCFβTrCP-EMI1-APC/C axis has been proven to play critical role of in genome reduplication [29,30]. Our findings provided evidence that the SCFβTrCP-EMI1-APC/C axis is present in the mouse oocyte and plays a crucial role in regulating mouse oocyte meiotic maturation. "
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    ABSTRACT: RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCF(βTrCP)-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation.
    Full-text · Article · Jul 2013 · PLoS ONE
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    • "For PLK1 knockdown, cells were transfected with a siRNA targeting the sequence 5′-AGAUUGUGCCUAAGUCUCU-3′ [69,70]. Transfection of siRNAs was carried out using DharmaFECT reagent (Thermo Scientific, Dharmacon) according to manufacturer’s instructions. "
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    ABSTRACT: Background Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. Results Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2−/− HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis. Conclusion Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis.
    Full-text · Article · May 2013 · Cell Division
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    • "Even in the absence of transcription factor E2F activation and basal levels of cyclin A transcription, Emi1's overexpression is apparently sufficient to drive S phase by stabilizing cyclin A [18]. Before mitosis , at the G1/S phase and throughout the S phase, Evi5 binds to a site adjacent to Emi1's degron, blocking degron phosphorylation by Polo-like kinases (Plks) [4], particularly Plk1 [19], thereby allowing the accumulation of Cyclin A for the progression of S phase [20]. Upon the onset of mitosis, phosphorylation of Emi1 leads to degradation by the Skp, Cullin, F-box (Fb) containing protein (SCF) E3 ubiquitin ligase complex, one of which consists of a b-transducin repeat containing protein (bTrCP)/Fbw1a [21]. "
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    ABSTRACT: The Ecotropic viral integration site 5 (Evi5) and Evi5-like (Evi5L) belong to a small subfamily of the Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins with enigmatically divergent roles as modulators of cell cycle progression, cytokinesis, and cellular membrane traffic. First recognized as a potential oncogene and a cell cycle regulator, Evi5 acts as a GTPase Activating Protein (GAP) for Rab11 in cytokinesis. On the other hand, its homologue Evi5L has Rab-GAP activity towards Rab10 as well as Rab23, and has been implicated in primary cilia formation. Recent genetic susceptibility analysis points to Evi5 as an important factor in susceptibility to multiple sclerosis. We discuss below the myriad of cellular functions exhibited by the Evi5 family members, and their associations with disease conditions.
    Preview · Article · May 2013 · FEBS letters
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