Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

Abstract

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells. INTRODUCTIONIn eukaryotes, the cell division cycle is regulated by the periodic activation and inactivation of cyclin-dependent kinases (Cdk), which are thought to phosphorylate protein substrates that are involved in driving cell cycle transitions (reviewed in Sherr, 1996;Roberts, 1999). The transition from G1 to S phase of the mammalian cell cycle requires the activity of cyclin E/Cdk2 and cyclin A/Cdk2. Cyclin E is expressed in middle to late G1 (Dulic et al., 1992, Koff et al., 1992, whereas cyclin A is first expressed at the G1/S transition of the cell cycle (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992). Microinjection of anticyclin E or anticyclin A antibodies into human cells or expression of antisense cyclin A RNA, inhibits initiation of DNA replication (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Tsai et al., 1993;Ohtsubo et al., 1995). Furthermore, enhanced levels of cyclins A and E accelerate the G1/S transition in vivo (Resnitzky et al., 1994(Resnitzky et al., , 1995 and in vitro (Krude et al., 1997).The initiation of DNA replication in eukaryotes requires the stepwise assembly of a multiprotein complex, called the prereplicative complex (pre-RC), on replicator elements in the chromatin (reviewed in Dutta and Bell, 1997;Newlon, 1997). Studies in yeast and in Xenopus have determined that a six-subunit complex, called origin recognition complex (ORC), serves to nucleate this assembly. In G1 of the cell cycle, the Cdc6 protein promotes the loading of the minichromosome maintenance proteins (MCMs) onto chromatin (Coleman et al., 1996;Aparicio et al., 1997;Donovan et al., 1997;Tanaka et al., 1997). This reaction requires an intact Walker A or Walker B motif in Cdc6 (Perkins and Diffley, 1998;Wang et al., 1999;Weinreich et al., 1999) and presumably involves direct physical interaction between Cdc6 and ORC (Li and Herskowitz, 1993;Liang et al., 1995;Mizushima et al., 2000). Although the mechanism of pre-RC assembly in human cells has not yet been characterized, all of the components of the pre-RC identified so far in yeast appear to be conserved in humans.A growing body of evidence indicates that components of the pre-RC are Cdk substrates who...

Analysis of Cdc6 function in the assembly of mammalian prereplication complexes

“…Human Cdc6 contains three consensus cdk phosphorylation sites within an amino terminal domain, and several studies have demonstrated that phosphorylation of Cdc6, most likely by cyclin A͞Cdk2, leads to its export from the nucleus to the cytoplasm, presumably to prevent reinitiation of replication in G 2 (19)(20)(21). In addition, Herbig et al (39) and Jiang et al (21) also have suggested that phosphorylation of Cdc6 may play a positive role in Cdc6 function because overproduction or microinjection of a mutationally altered Cdc6 that cannot be phosphorylated by cdks blocked DNA replication. In contrast, Petersen et al (20) and Pelizon et al (19) performed similar experiments but observed no consequences of the lack of Cdc6 phosphorylation other than a failure to relocalize Cdc6 during S phase.…”

“…We also provide evidence that phosphorylation of Cdc6 is not required for Mcm chromatin loading. Previous studies have offered conflicting data on this point with some investigators reporting no consequences (19,20) whereas others report a dominant interfering effect and suggest that phosphorylation of Cdc6 is required for origin firing (21,39). Possible explanations for the discrepancies between these studies include differences in the form of Cdc6 that is overproduced, particularly when it is expressed as a fusion protein, or the extent to which Cdc6 is overexpressed.…”

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

“…It is well established that the stability of Cdc6 protein is regulated by phosphorylation at 3 canonical CDK sites within its N-terminal domain, 8,9 which depends on acetylation by the acetyltransferase GCN5 at specific residues nearby. 10 In quiescent mammalian cells the anaphase promoting complex (APC) E3 ubiquitin ligase provides for constant proteasomal degradation of Cdc6.…”

Down-regulation of Cdc6, a Cell Cycle Regulatory Gene, in Prostate Cancer

“…In middle to late G 1 and at the G 1 /S transition, S-phase cyclins (cyclin E and cyclin A) and cdk2 are expressed (21,22) and initiate DNA replication by phosphorylating CDC6, which causes it to dissociate from Pre-RC (23). Release of CDC6 from chromatin and its translocation to the cytoplasm triggers initiation of DNA replication (24). Another role of CDC6 in S. cerevisiae is to function as an inhibitor of the G 2 /M phase until the S phase is complete (25).…”