Chan A K, Persad S, Litchfield D W, Wright J A
Manitoba Institute of Cell Biology, Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada.
Biochim Biophys Acta. 1999 Jan 11;1448(3):363-71. doi: 10.1016/s0167-4889(98)00115-3.
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 p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 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 p34cdc2 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 p34cdc2 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 p34cdc2 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.
核糖核苷酸还原酶是DNA合成中的一种限速酶，由两种不同的蛋白质R1和R2组成。R2蛋白似乎是增殖细胞中酶活性的限速因子，它被p34cdc2和CDK2磷酸化，这两种蛋白是细胞周期转换事件的介质。R2蛋白中丝氨酸20处的序列与p34cdc2和CDK2激酶的共有序列相匹配。我们测试了丝氨酸20残基是p34cdc2激酶主要磷酸化位点的假说。合成了三种肽（从Asp-13到Ala-28），它们分别包含野生型氨基酸序列（Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala，丝氨酸肽）或一种突变体，其中丝氨酸残基被丙氨酸残基（丙氨酸肽）或苏氨酸残基（苏氨酸肽）取代。只有丝氨酸肽和苏氨酸肽被p34cdc2激酶磷酸化。在丝氨酸肽和天冬氨酸-N端蛋白酶消化的R2蛋白的二维磷酸肽图谱实验中，肽的共迁移模式表明，含有丝氨酸20的合成磷酸肽与主要的天冬氨酸-N消化的R2磷酸肽相同。为了进一步测试丝氨酸20是R2蛋白上主要磷酸化残基的假说，通过定点诱变产生了三种重组R2蛋白（R2-Thr、R2-Asp和R2-Ala），其中丝氨酸20残基被苏氨酸、天冬氨酸或丙氨酸残基取代。野生型R2和苏氨酸取代的R2蛋白（R2-Thr）被p34cdc2激酶磷酸化，而在相同实验条件下，未检测到R2-Asp和R2-Ala的磷酸化。此外，R2-Thr蛋白中的磷酸化氨基酸残基被确定为磷酸苏氨酸。因此，通过将丝氨酸20残基替换为苏氨酸，R2蛋白中的磷酸化氨基酸变为磷酸苏氨酸。总的来说，这些结果确凿地证实，核糖核苷酸还原酶R2蛋白上一个主要的p34cdc2磷酸化位点位于N末端附近的丝氨酸20处，该位点位于序列Ser-Pro-Leu-Lys-Arg-Leu中。野生型和突变型R2蛋白之间核糖核苷酸还原酶活性的比较表明，丝氨酸20处的突变对酶活性没有显著影响。
