FIGURE 5.

DDK phosphorylates Ser-164 and Ser-170 of Mcm2. A, residues 161–181 of S. cerevisiae Mcm2 are shown, and Ser-164 and Ser-170 were targeted for site-directed mutagenesis. B, mutants of Mcm2-(1–278) of varying amounts were incubated with 50 ng of DDK and [γ-³²P]ATP in a volume of 10 μl for 30 min at 30 °C. The amount of Mcm2-(1–278) added was 12 or 4 pmol (left image) or 1.2 or 0.4 pmol (right image). The reactions were then analyzed by SDS-PAGE followed by phosphorimaging. C, results from experiments similar to B were quantified, and the fraction of phosphate incorporation is plotted as a function of input in picomoles. The data are mean ± S.E. D, mutants of Mcm2-(1–278) with a site for protein kinase A phosphorylation were radiolabeled with [γ-³²P]ATP and protein kinase A (PKA) as described under “Experimental Procedures.” 50 pmol of GST-Dbf4 was incubated with varying amounts of radiolabeled Mcm2 fragments for 1 h at room temperature in a total volume of 100 μl. After mixing, the reactions were analyzed as described under “Experimental Procedures.” E, sequence alignment of budding yeast Mcm2 and budding yeast Mcm4. Serine and threonine residues are in boldface, and acidic residues are underlined. The aligned regions of these two proteins contain phosphoacceptor sites for DDK. F, alignment of budding yeast Mcm2 with Mcm2 from higher eukaryotes. DDK phosphoacceptor sites of human Mcm2 do not map to this region (10, 19, 20).