RESUMO
Anaphase onset is an irreversible cell cycle transition that is triggered by the activation of the protease Separase. Separase cleaves the Mcd1 (also known as Scc1) subunit of Cohesin, a complex of proteins that physically links sister chromatids, triggering sister chromatid separation. Separase is regulated by the degradation of the anaphase inhibitor Securin which liberates Separase from inhibitory Securin/Separase complexes. In many organisms, Securin is not essential suggesting that Separase is regulated by additional mechanisms. In this work, we show that in budding yeast Cdk1 activates Separase (Esp1 in yeast) through phosphorylation to trigger anaphase onset. Esp1 activation is opposed by protein phosphatase 2A associated with its regulatory subunit Cdc55 (PP2ACdc55) and the spindle protein Slk19. Premature anaphase spindle elongation occurs when Securin (Pds1 in yeast) is inducibly degraded in cells that also contain phospho-mimetic mutations in ESP1, or deletion of CDC55 or SLK19. This striking phenotype is accompanied by advanced degradation of Mcd1, disruption of pericentric Cohesin organization and chromosome mis-segregation. Our findings suggest that PP2ACdc55 and Slk19 function redundantly with Pds1 to inhibit Esp1 within pericentric chromatin, and both Pds1 degradation and Cdk1-dependent phosphorylation of Esp1 act together to trigger anaphase onset.
Assuntos
Anáfase/fisiologia , Proteína Quinase CDC2/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Separase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Mutação , Fosforilação , Proteína Fosfatase 2/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Securina/genética , Securina/metabolismo , Separase/genética , Fuso Acromático/genética , CoesinasRESUMO
In cell culture, many adherent mammalian cells undergo substantial actin cytoskeleton rearrangement prior to mitosis as they detach from the extracellular matrix and become spherical. At the end of mitosis, the actin cytoskeleton is required for cytokinesis and the reassembly of interphase structures as cells spread and reattach to substrate. To understand the processes regulating mitotic cytoskeletal remodeling, we studied how mitotic phosphorylation regulates filamin A (FLNa). FLNa is an actin-crosslinking protein that was previously identified as a cyclin-dependent kinase 1 (Cdk1) binding partner and substrate in vitro. Using quantitative label-based mass spectrometry, we find that FLNa serines 1084, 1459 and 1533 are phosphorylated in mitotic HeLa cells and all three sites match the phosphorylation consensus sequence of Cdk1. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine residues and expressed GFP-tagged FLNa(S1084A,S1459A,S1533A) (FLNa-AAA GFP) in a FLNa-deficient human melanoma cell line called M2. M2 cells expressing FLNa-AAA GFP have enhanced FLNa-AAA GFP and actin localization at sites of contact between daughter cells, impaired post-mitotic daughter cell separation and defects in cell migration. Therefore, mitotic phosphorylation of FLNa is important for successful cell division and interphase cell behavior.