Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

Abstract

BackgroundAlthough the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis.Methodology/Principal FindingsMitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell.ConclusionsCollectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1.

Cyclin-dependent Kinase 1-dependent Phosphorylation of cAMP Response Element-binding Protein Decreases Chromatin Occupancy

“…We note that CDK1-dependent phosphorylation of AB1 and TFII-I promoted their chromatin eviction during mitosis (47,48). Additionally, the C2H2 zinc finger DNA binding domain transcription factors were phosphorylated and inactivated during mitosis (46).…”

ATF7 is stabilized during mitosis in a CDK1-dependent manner and contributes to cyclin D1 expression

“…31 AIB1, a transcriptional co-activator, which promotes pre-neoplastic changes and cancer initiation in animal models, is phosphorylated on Ser728 in mitotic cells in a CDK1-dependent way, resulting in its exclusion from the condensed chromatin. 32 However, neither did phosphorylation nor point mutations of the Ser728 into alanine or glutamate impair its recruitment onto a minimal promoter and capacity to co-activate its target genes during mitosis. 32 These results highlight the difficulties to establish a strict causality between phosphorylation events and displacement of transcription factors from the condensed chromatin in a cellular context.…”

“…32 However, neither did phosphorylation nor point mutations of the Ser728 into alanine or glutamate impair its recruitment onto a minimal promoter and capacity to co-activate its target genes during mitosis. 32 These results highlight the difficulties to establish a strict causality between phosphorylation events and displacement of transcription factors from the condensed chromatin in a cellular context.…”

“…For radioactive reactions, MBP (SigmaAldrich) was incubated in the same conditions with the respective kinase and [g- 32 P] ATP to a final specific activity of 150 mCi /mmol. Proteins were analyzed by western blot or exposed to a phosphoimager screen and scanned with a Typhoon FLA9500.…”

Protein Phosphatases Drive Mitotic Exit

“…Finally, PP1 appears to play a role in the initiation of transcription upon entry into the next cell cycle, as it is responsible for reversing the inhibitory Cdk1-mediated phosphorylation events of the transcription factor, AIB1 (Ferrero et al, 2011). AIB1 phosphorylation does not appear to affect its transcriptional activity but instead excludes it from condensed chromatin during mitosis to prevent its access to the promoters of AIB1-dependent genes.…”