Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.
The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.
Krek
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Swiss Institute for Experimental Cancer Research, ISREC, Epalinges.
Nigg
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001358:Avian Sarcoma Viruses / Q000235:genetics; D016203:CDC2 Protein Kinase / Q000276:immunology / Q000378:metabolism*; D002453:Cell Cycle; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D016193:G1 Phase; D016195:G2 Phase; D007118:Immunoassay; D007700:Kinetics; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D016196:S Phase
D000906:Antibodies; D010748:Phosphopeptides; D016203:CDC2 Protein Kinase