CDC 6 controls dynamics of the fi rst embryonic M-phase entry and progression via CDK 1 inhibition

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle. & 2014 Elsevier Inc. All rights reserved. Introduction CDC6, or Cell Division Cycle 6 protein, a member of the family of AAAþATPases, is an essential and highly regulated component of the pre-replication complex required for loading MiniChromosome Maintenance (MCM) proteins onto the DNA (Perkins and Diffley, 1998; Weinreich et al., 1999). In the majority of organisms only one isoform of this protein is present. Mus musculus is the only organism, in which the presence of two isoforms of this protein was reported. Mouse isoforms of CDC6 are different variants of transcripts, which most probably appeared as result of alternative splicing. Variant 1 of cdc6 gene transcript (# NM 011799.2 GenBank) was localized for the first time in mouse Embryonic Stem Cells (ES; Berger et al., 1999). Variant 2 was identified in the brain tissues of mouse embryo at 12.5 day of development (Strausberg et al., 2002). Variant 1 encodes isoform a, and variant 2 isoform b of Cdc6 protein. Both isoforms are highly homologous. In contrast to its function during DNA synthesis the role of CDC6 in mitosis is much less understood. It was shown that in yeast, CDC6 plays a dual role in the cell cycle control and it’s over expression delays the M-phase entry (Bueno and Russell, 1992). CDC6 interacts with CDK1 upon Mphase exit; binding of CDC6 to CDK1 inhibits its kinase activity participating to exit frommitosis. Deletion of CDC6 lacking the CDKinteracting domain has no effect on DNA replication during S-phase, but slows down the mitotic exit (Elsasser et al., 1996; Calzada et al., 2001). However, these results were challenged by the experiments showing that in yeast exit from mitosis occurs in the absence of CDC6 (Archambault et al., 2003). In HeLa cells CDC6 is phosphorylated during the M-phase by Polo-like kinase 1 (Plk1) (Yim and Erikson, 2010). CDC6 associates with Plk1 and localizes at the spindle pole in metaphase and central spindle in anaphase. The increase in CDC6 phosphorylation correlates with the increased level of Plk1; conversely CDC6 is hypophosphorylated in Plk1-depleted cells. Phosphorylation of CDC6 by Plk1 is required for its interaction with CDK1 and for CDK1 inhibition