ACTIVATION OF P34(CDC2) PROTEIN-KINASE BY MICROINJECTION OF HUMAN CDC25C INTO MAMMALIAN-CELLS - REQUIREMENT FOR PRIOR PHOSPHORYLATION OF CDC25C BY P34(CDC2) AT MITOSIS ON SITES PHOSPHORYLATED

Human cdc25C protein, a specific tyrosine phosphatase that activates t he p34(cdc2) protein kinase at mitosis, is itself a phosphoprotein tha t shows increased phosphorylation during the G(2)-M transition. In vit ro, cdc25C protein is substantially phosphorylated by purified p34(cdc 2)-cyclin B protein kinase. Of seven putative phosphorylation sites fo r p34(cdc2) protein kinase present in human cdc25C, five are phosphory lated by p34(cdc2) protein kinase in vitro, as assessed by tryptic pho sphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G(2)-M transition in normal mammalia n fibroblasts and have been precisely mapped. The cdc25C phosphorylate d in vitro by p34(cdc2) protein kinase exhibits a 2-3-fold higher acti vity than the nonphosphorylated cdc25C, as assayed by activation of in active cdca prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G(2) cells into premature prophase in a manner similar to microinjection of purified active p34(cdc2) pro tein kinase. Together these data show that multisite phosphorylation o f cdc25C by p34(cdc2)-cyclin B protein kinase occurs at the G(2)-M tra nsition and is sufficient to induce the autoamplification of cdc2/M-ph ase promoting factor necessary to drive somatic mammalian cells into m itosis.