Regulation of mitotic spindle biogenesis in budding yeast.
Assembly of the mitotic spindle is critically dependent on centrosome duplication (spindle pole body or SPB in yeast), separation of sister centrosomes and microtubule dynamics. Activation of Cdc28 (Cdk1) by tyrosine-19 dephosphorylation is essential for centrosome separation, the first step in bipolar spindle formation. In this study, we have investigated the regulatory role of Cdk1 in centrosome separation. We show that E3 ubiquitin ligase APCCdh1 (conserved from yeast to human) acts as a potent inhibitor of spindle assembly by promoting degradation of microtubule-associated proteins essential for centrosome separation. Activated Cdk1 causes inactivation of APCCdh1 during S phase, resulting in the accumulation of centrosome-separation-promoting proteins. Phosphorylation of Cdh1 by Cdk1 creates a binding site for Polo kinase (Cdc5) which leads to further phosphorylation of Cdh1 by polo kinase. Hence, synergistic action of Cdk1 and Polo kinase is required for complete inactivation of Cdh1 (and thus, spindle assembly) in which Cdk1 acts as a priming-kinase for the Polo kinase. We further show that Polo kinase becomes essential for centrosome separation in the absence of Acm1, a negative regulator of APCCdh1 whose action is independent of the status of Cdh1 phosphorylation. Hence, we have uncovered a novel control circuit (involving Cdk1, Cdh1, Polo kinase and microtubule associated proteins) that regulates centrosome separation and the initiation of spindle assembly.
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