Site‐specific phosphorylation of MCM4 during the cell cycle in mammalian cells

Abstract

MCM2-7 proteins are essential for eukaryotic DNA replication and are the most likely candidates for the replicative DNA helicase responsible for unwinding DNA at the replication forks [1][2][3]. Consistent with their primary amino acid sequences, a subcomplex of MCM4 ⁄ 6 ⁄ 7 functions as DNA helicase in vitro [4]. It has been suggested that MCM2, -3 and -5 play a regulatory role in the function of MCM4 ⁄ 6 ⁄ 7 DNA helicase, because addition of MCM2 or MCM3 ⁄ 5 to MCM4 ⁄ 6 ⁄ 7 complex resulted in inhibition of the MCM4 ⁄ 6 ⁄ 7 DNA helicase [5,6]. Thus MCM2-7 complex, a major MCM complex on chromatin during the G 1 phase, has to be activated to show DNA helicase activity. It is possible that several proteins, including CDC7 kinase and CDC45 are involved in this activation. Evidence suggests that MCM2-7 proteins may have additional functions during the cell cycle [3]. Cyclin-dependent kinases (CDK), which play a critical MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G 2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2 ⁄ cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.Abbreviations CDK, cyclin-dependent kinases.

Identification and Functional Analysis of a Novel Cyclin E/Cdk2 Substrate Ankrd17

“…It has been found that phosphorylation of MCM family members by cyclin/Cdks regulates their assembly into the prereplication complex and their helicase activity, which is essential for DNA replication (30,48,49). Ankrd17 can be phosphorylated by cyclin E/Cdk2, but whether phosphorylation of Ankrd17 regulates its association with the components in the pre-replication complex and loading onto chromatin and controls the activity of pre-RC needs to be further elucidated.…”

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

“…However, the demonstration of an essential role for these putative phosphorylation events has been elusive for several reasons: (i) the only essential targets of CDK phosphorylation in S. cerevisiae are the replication factors Sld2 and Sld3, as the appropriate genetic manipulation of these factors obviates the normal dependence of DNA replication on CDK phosphorylation (247,279); (ii) treatment of purified Mcm2-7 with either phosphatase (to completely dephosphorylate the Mcm proteins) or Cdc28/Clb5 (the yeast CDK1 homologue) has no obvious effect on the in vitro ability of the complex to bind DNA (Bochman, unpublished); and (iii) the ablation of all consensus or near-consensus CDK and ATR phosphorylation sites (ϳ60 total, alanine substitution mutations of the critical serine/ threonine) from the six S. cerevisiae MCM genes demonstrates that, with the exception of a site within the Walker A motif of all six subunits, none of these phosphorylation sites on any one Mcm subunit are essential for viability (M. Patel, J Mitchell, D. Leigley, R. Elbakri, and A. Schwacha, unpublished observations). These results can be interpreted in one of two ways: that CDK and ATR phosphorylation of Mcm2-7 has little or no importance in DNA replication or, conversely, that it is so important that there exists a high level of functional redundancy between phosphorylation sites that has so far masked current analyses, a possibility that has been demonstrated in a different context (189 [179]) and suggests that these modifications may have distinct and site-specific roles (133). In vitro, CDK phosphorylation of human Mcm4 Thr-19 and -110 decreases Mcm467 helicase activity (111).…”

DNA Replication in the Archaea

“…The N termini of the proteins are less well conserved and are thought to be responsible for multimerization and regulation (30,33). In eukaryotic MCM proteins, the phosphorylation sites for cyclin-dependent kinases and other regulatory kinases are mostly located in this region (54,86). There is also a helix-turn-helix domain at the extreme C terminus of the protein.…”