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 cyclin-dependent and Cdc7 kinases is essential for the initiation of DNA replication.

Fig. 1.

Phosphorylation of the N terminus at a region of human Mcm2 protein by Cdc7 kinase. GST-tagged Mcm2 truncated proteins (0.2 μg, each equivalent to ≈5 pmol of hM2-N1 or hM2-N2, and 2 pmol of hM2-C) were incubated with indicated amounts of Cdc7 kinase (+, 5 ng; ++, 15 ng) in the kinase assay reaction, as described in Materials and Methods, at 30°C for 30 min. Mixtures were then subjected to SDS/PAGE followed by staining with Coomassie blue (Upper) and autoradiography (Lower). The numbers at the bottom of the autoradiogram indicate the ³²P incorporation, determined by PhosphoImager analysis. The symbol – at the bottom of the autoradiogram denotes incorporation of <0.01 pmol. hM2-N1, GST fusion protein containing the amino acid 1–70 region of human Mcm2 protein; hM2-N2, amino acids 98–180; hM2-C, amino acids 175–904.

Fig. 2.

Phosphorylation of GST-tagged Mcm2 N-terminal peptides. (A) Amino acid sequence of the Mcm2 N terminus and peptide regions fused to GST. (B) GST-tagged Mcm2 N-terminal peptides (A–F, 1 μg, ≈30 pmol) or full-length Mcm2 protein (Full, 0.3 μg, 3 pmol) were phosphorylated with Cdc7 kinase (+, 5 ng; ++, 15 ng) at 30°C for 30 min. The mixtures were subjected to SDS/PAGE followed by staining with Coomassie blue (Upper) and autoradiography (Lower).

Fig. 3.

Identification of phosphorylation sites in the A peptide region by alanine substitution analysis. (A) The amino acid sequences of several alanine-substituted peptides of the A region that were fused to GST protein. (B and C) GST-tagged Mcm2 N-terminal peptides (3 μg, 90 pmol) were incubated with Cdc7 kinase (+, 5 ng; ++, 15 ng) at 30°C for 15 min. The mixtures were subjected to SDS/PAGE followed by staining with Coomassie blue and autoradiography.

Fig. 4.

The ⁵S and ⁵³S residues of Mcm2 protein are major phosphorylation sites. The full-length and alanine-substituted Mcm2 proteins, as indicated, were expressed and purified by using the E. coli expression system. Mcm2 proteins (0.15 μg, 1.5 pmol) were incubated with indicated amounts of Cdc7 kinase at 30°C for 15 min. The mixtures were then subjected to SDS/PAGE followed by staining with Coomassie blue and autoradiography. The symbol – at the bottom of the autoradiogram denotes the incorporation of <1 fmol.

Fig. 5.

Influence of the acidic amino acids bordering phosphorylation sites on Cdc7 kinase activity. (A) Amino acid sequences of the modified GST-tagged hM2E peptides. (B and C) GST-tagged Mcm2 N-terminal peptides (1 μg, 30 pmol) were incubated with Cdc7 kinase (+, 1 ng; ++, 3 ng) at 30°C for 20 min and mixtures subjected to SDS/PAGE followed by staining with Coomassie blue and autoradiography.

Fig. 6.

The generation of additional Cdc7 phosphorylation sites by prior phosphorylation. (A) The amino acid sequences of two synthetic peptides with or without the phosphor-serine at the CDK phosphorylation site. (B) The two hM2B oligopeptides were synthesized chemically (Peptron). These oligopeptides (5 μg, 3.8 nmol) were incubated with the indicated levels of Cdc7 kinase at 30°C for 20 min. Reaction mixtures were then subjected to 18% Tricine-SDS/PAGE followed by autoradiography. The symbol –, located at the bottom of the autoradiogram, indicates that incorporation was <0.1 pmol.