CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein

Abstract

Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ( 5 S and 53 S) and at least three minor phosphorylation sites ( 4 S, 7 S, and 59 T) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of 5 S and 53 S by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19 -30 and containing 26 S and 27 S), it phosphorylated 26 S efficiently when this peptide contained a chemically synthesized phospho-27 S modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclindependent and Cdc7 kinases is essential for the initiation of DNA replication.replication ͉ prereplicative complex ͉ origin activation C dc7 plays an essential role in the initiation of eukaryotic DNA replication (1-3). Cdc7 encodes a serine͞threonine kinase that is highly conserved from yeast to human (4). The activity of this kinase fluctuates during the cell cycle and depends completely on the regulatory subunit, Dbf4. Dbf4 accumulates during the S and G 2 phases of the cell cycle and is degraded rapidly during the G 1 phase by the anaphase promoting complex (5, 6). In Saccharomyces cerevisiae, Dbf4 binds to chromatin at the G 1 ͞S transition and remains on chromatin during the S phase (7). In the Xenopus egg extract cell-free system, Cdc7 was found to bind to chromatin during S phase, and this association required the minichromosome maintenance (MCM) complex (8).Both genetic and biochemical studies in S. cerevisiae indicate that Cdc7 kinase activity is required throughout S phase to activate replication origins on chromosomes (9). Cdc7 kinase acts directly on individual replication origins, presumably by phosphorylating components of the prereplicative complex (pre-RC), which may lead to the remodeling of the pre-RC for the unwinding of replication origins and subsequent recruitment of the replication fork machinery. Although the mechanism-oforigin activation by Cdc7 kinase remains unclear, the MCM complex appears to be the primary physiological target of this kinase activity (2).The MCM complex is composed of six distinct subunits (Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, and Mcm7), which are all related structurally and highly conserved in eukaryotes (10). All six proteins are essential for the initiation of DNA replication and contain sequence motifs required for DNA helicase activity. In keeping with this notion, the MCM subcomplex containing Mcm4, Mcm6, and Mcm7 posses...

Incorporation into the prereplicative complex activates the Mcm2–7 helicase for Cdc7–Dbf4 phosphorylation

“…Does the priming phosphorylation play a role in DDK target peptide recognition? This possibility is supported by several studies suggesting that DDK can target serines or threonines adjacent to phosphoserine or phosphothreonine (Cho et al 2006;Masai et al 2006;Montagnoli et al 2006;Wan et al 2008). Indeed, a perusal of the sequences of the N terminus of Mcm4 and Mcm6 shows a large number of clusters of Ser/Thr.…”

“…Previous studies have shown that DDK phosphorylation of Mcm2 is dramatically increased when it has been previously phosphorylated (Kihara et al 2000;Cho et al 2006;Montagnoli et al 2006). Furthermore, several Mcm2-7 subunits have been shown to be phosphoproteins in G1 cells in vivo Li et al 2007).…”

“…In vitro assays have shown that DDK can phosphorylate multiple subunits of the Mcm2-7 complex (Weinreich and Stillman 1999;Kihara et al 2000), Cdc45 (Nougarede et al 2000), and DNA Pol a (Weinreich and Stillman 1999). Of these proteins, both in vivo and in vitro studies suggest that Mcm2-7 subunits are most likely to be the essential DDK targets (Hardy et al 1997;Lei et al 1997;Masai and Arai 2002;Cho et al 2006;Masai et al 2006). DDK activity is required for robust association of Cdc45 with chromatin (Walter 2000;Zou and Stillman 2000), and recent studies suggest that Cdc45 and the four-protein GINS complex associate with and activate the helicase activity of the Mcm2-7 complex (Gambus et al 2006;Moyer et al 2006;Pacek et al 2006).…”

How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

“…As with the yeast protein, vertebrate Cdc7 has a strong preference for Ser or Thr in an acidic context, either produced by adjacent amino acids that are acidic, or else due to an adjacent CDK site that appears to prime phosphorylation by Cdc7 (Masai et al 2000(Masai et al , 2006Cho et al 2006;Montagnoli et al 2006). In the case of Mcm4, isolated fragments from the N-terminal tail are phosphorylated less well than full-length protein, perhaps indicating the presence of a ''docking domain'' analogous to that of budding yeast Mcm4 (Masai et al 2006).…”

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation

“…The DDKs phosphorylate multiple subunits of the Mcm2-7 complex to promote activation of the helicase (Tanaka and Araki 2013). MCM phosphorylation by DDKs is highly conserved in eukaryotes, but it appears that the mechanism by which CDKs stimulate initiation in higher eukaryotes has diverged significantly (Masai et al 2000(Masai et al , 2006Cho et al 2006;Montagnoli et al 2006). Most of what is known about the function of CDK in initiation is from studies in yeast.…”