Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle.
RP-A is a single-stranded DNA-binding protein, which has been shown to be required for DNA replication using an SV40 model system. The protein has also been shown to be phosphorylated at the G1-S phase transition. Using Xenopus cell-free extracts we have investigated the role of RP-A in nuclear replication and characterized the kinases and conditions that lead to phosphorylation of RP-A during the cell cycle. By immunodepleting RP-A from Xenopus extracts we have shown that RP-A is essential for replication of chromosomal DNA. Our results show that, during S phase, only that RP-A which is associated with nuclei is phosphorylated. Furthermore our results indicate that during S phase RP-A is only phosphorylated when associated with single-stranded DNA. By immunodepleting cdk2 kinase we show that cdk2 kinase is required for the observed phosphorylation of RP-A in nuclei during S phase. However, using purified cdk2 kinase and RP-A we are unable to detect a direct phosphorylation of RP-A by cdk2 kinase. This observation suggests that phosphorylation of DNA-bound RP-A at S phase is carried out by a kinase distinct from cdk2. Consistent with this we find that when single-stranded DNA is added to S phase extracts depleted of cdk2 kinase, RP-A is phosphorylated. Together these results suggest that cdk2 kinase participates in the activation of DNA replication at a stage prior to the binding of RP-A to the initiation complex. In addition to RP-A phosphorylation in S phase, we have also found that at the onset of mitosis RP-A is quantitatively phosphorylated and that phosphorylation is directly mediated by cdc2 kinase. However, at this time during the cell cycle, cdc2-dependent phosphorylation of RP-A is independent of DNA binding. These observations further demonstrate the distinctions between cdk2 and cdc2 kinases.
Fang
F
F
Department of Biology, University of California, San Diego, La Jolla 92093-0322.
Newport
J W
JW
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology*; D002474:Cell-Free System; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051716:Replication Protein A; D016196:S Phase / Q000502:physiology; D014981:Xenopus; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D051716:Replication Protein A; D029867:Xenopus Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases