CELL-CYCLE REGULATION OF THE P34(CDC2) INHIBITORY KINASES

In cells of higher eukaryotic organisms the activity of the p34(cdc2)/ cyclin B complex is inhibited by phosphorylation of p34(cdc2) at two s ites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosp horylating p34(cdc2) On Thr14 and Tyr15 was examined in extracts prepa red from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- spec ific kinase activities were regulated in a cell cycle-dependent manner . The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15- specific k inase activities remained high, suggesting that a decrease in Thr14- a nd Tyr15- kinase activities may be required for entry of cells into mi tosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activat ion of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but bp the balance between Thr14/ Tyr13 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34 on Thr14 and/or Tyr15 in in terphase extracts prepared from Xenopus eggs. An activity capable of p hosphorylating Tyr15 remained soluble after ultracentrifugation of int erphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activit ies that were detergent extractable and that phosphorylated p34(cdc2) on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activiti es co-purified through three successive chromatographic steps indicati ng the presence of a dual-specificity protein kinase capable of acting on p34(cdc2).