Inactivation of Cdh1 by synergistic action of Cdk1 and polo kinase is necessary for proper assembly of the mitotic spindle

Abstract

Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase, which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.Centrosomes function as microtubule organizing centres (MTOC) and are responsible for the bipolar nature of the mitotic spindle 1 . Misregulation of the centrosome cycle can produce an abnormal number of centrosomes and multi-polar spindles, and cause chromosome missegregation resulting in aneuploidy. Centrosomal abnormalities and aneuploidy are common in many types of cancers 2 .In mammalian cells, centrosomes consist of a pair of centrioles and the surrounding dense fibrillar mass known as the pericentriolar material 3 . A cell inherits one centrosome from its progenitor but replicates it as it progresses through the cell cycle. The duplicated centrosomes remain tethered to each other by a linker until the time of mitotic entry, when the linkage between the sister centrosomes is severed and they move away from each other, eventually positioning themselves face-to-face, separated by an interdigitated array of microtubules. The structure and duplication of centrosomes have been studied in some detail; however, the process by which they separate is not well understood.3Correspondence should be addressed to U.S. (e-mail: E-mail: mcbucs@imcb.a-star.edu.sg). Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONS K.C. and L.H.H. performed all experiments; T.H.G. and M.W. carried out the electron microscopy analysis; U.S. and K.C. planned the project and analysed the data. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access The spindle pole body (SPB), a multi-protein assembly, is the centrosome-equivalent in S. cerevisiae 4 . Yeast daughter cells inherit from the mother cells one SPB bearing a half-bridge (a structure that extends from one side of the SPB) and duplicate it as they traverse START (the point in late G1 that precedes DNA replication). The bridge between the duplicated SPBs is severed in late S pha...

Inhibitory Phosphorylation of Cyclin-Dependent Kinase 1 as a Compensatory Mechanism for Mitosis Exit

“…APC/C also binds another targeting subunit called CDH1. In marked contrast to CDC20, phosphorylation of CDH1 by cyclin B1-CDK1 prevents its binding to APC/C, thereby keeping APC/C CDH1 inactive during mitosis (8). The inactivation of CDK1 at the end of mitosis relieves the inhibition of APC/ C CDH1 (reviewed in reference 30).…”

The Aurora kinase Ipl1 is necessary for spindle pole body cohesion during budding yeast meiosis

“…The Polo kinase, Cdc5 in yeast, is implicated in SPB separation in vegetative yeast cells and centriole separation in animal cells (Crasta et al, 2008;Tsou et al, 2009). Cdc5, C-terminally tagged with GFP, appeared in the nucleus ,20 minutes before MI SPB separation in wild-type cells, forming foci that were not associated with SPBs (Fig.…”

“…One of the major components of the satellite is the SPB core component, Spc42 (Donaldson and Kilmartin, 1996;Bullitt et al, 1997). Duplicated SPBs form a side-by-side configuration and are tethered together by the bridge, which is severed or disassembled upon the formation of a bipolar spindle, promoted by the actions of the mitotic cyclin Cdk and the Polo-like kinase Cdc5 (Kilmartin, 2003;Crasta et al, 2008). Two known structural components of the half bridge are Sfi1 and Cdc31, homologs of which are essential components in the vertebrate centriole (Li et al, 2006).…”

“Reductional anaphase” in replication-defective cells is caused by ubiquitin-conjugating enzyme Cdc34-mediated deregulation of the spindle

“…42 This is mainly due to stabilization of microtubule-associated proteins, such as Cin8, in the absence of Cdh1. 43 Therefore, we deleted CDH1 gene in cdc34-1 strain to allow the assembly of short, bipolar spindle and repeated the experiment. cdc34-1 cdh1Δ and cdc34-1 cdc6Δ MET-CDC6 cdh1Δ strains were synchronized in mitosis as described above, released in +met medium at 36°C, and the terminal phenotypes were monitored.…”

“…Among the upregulated proteins after the shift to 24°C, Cdc5 (yeast polo kinase) specifically drew our attention, since Cdc5 overexpression has been shown to induce spindle formation in spindle assembly-defective cdk1-as1 cells. 43 Further experiments showed that the ectopic expression of Cdc5 causes short spindle assembly in 30% of the cdc34-1 cells and induces long spindles in 60% of cdc34-1 cdh1Δ cells. Cdc5 pulse also becomes stabilized upon restoration of Cdc34 function by shifting Cdc34-1 cells from 36°C to 24°C (data not included).…”