Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34(cdc2) and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34(cdc2) and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34(cdc2) kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence(Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34(cdc2) kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34(cdc2) kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34(cdc2) phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity. (C) 1999 Elsevier Science B.V. All rights reserved.