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The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells

Authors
Kim, JS; Park, YY; Park, SY; Cho, H; Kang, D
Citation
The Journal of biological chemistry, 286(35):30615-30623, 2011
Journal Title
The Journal of biological chemistry
ISSN
0021-92581083-351X
Abstract
The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.
MeSH terms
Alanine/chemistryAnimalsBinding SitesCDC2 Protein Kinase/metabolismCell Cycle Proteins/*chemistry/*metabolismCyclin B1/metabolismFlow Cytometry/methodsG2 PhaseHeLa CellsHumansLysine/chemistry*MitosisModels, BiologicalNeoplasm Proteins/*chemistryProtein ConformationProtein-Serine-Threonine Kinases/*metabolismProto-Oncogene Proteins/*metabolismUbiquitin/*chemistryUbiquitin-Protein Ligases/*chemistry
DOI
10.1074/jbc.M111.231803
PMID
21768102
Appears in Collections:
Journal Papers > School of Medicine / Graduate School of Medicine > Biochemistry & Molecular Biology
AJOU Authors
박, 용예조, 혜성
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