Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.
Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.
Strausfeld
U
U
Department of Cell Biology, Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
Fernandez
A
A
Capony
J P
JP
Girard
F
F
Lautredou
N
N
Derancourt
J
J
Labbe
J C
JC
Lamb
N J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D016213:Cyclins / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008845:Microinjections; D008938:Mitosis; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D020687:cdc25 Phosphatases
D018797:Cell Cycle Proteins; D016213:Cyclins; D011506:Proteins; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases