DIFFERENTIAL REGULATION OF THE SYNTHESIS AND ACTIVITY OF THE MAJOR CYCLIN-DEPENDENT KINASES, P34(CDC2), P33(CDK2), AND P34(CDK4), DURING CELL-CYCLE ENTRY AND PROGRESSION IN NORMAL HUMAN T-LYMPHOCYTES

Three major cyclin-dependent kinases, p34(cdc2), P33(cdk2) and p34(cdk4) were examined in normal human T cells stimulated to enter the cell cycle in vitro. None of the three genes was expressed in resting T cells. Transcripts from the cdk4 and cdk2 genes were detectable as early as 3 and 8 hr after stimulation, respectively, whereas cdc2 gene transcripts were not detectable until about 24 hr, shortly before S phase entry. Immunoblot analysis showed that resting T cells contained little p34(cdk4), no p34(cdc2), and a low level of p33(cdk2) protein. Increased amounts of p34(cdk4), p33(cdk2) and p34(cdc2) proteins were seen at about 7, 10, and 30 hr after stimulation, respectively. Immunoprecipitates of each of the kinases were assessed for histone H1 kinase activity. Activity due to p33(cdk2) first became detectable in mid-G1 phase and increased dramatically after entry into S phase. Active p34(cdc2) kinase was not detected until about 40 hr after stimulation, about 10 hr after the first appearance of the protein. Immunoprecipitates of p34(cdk4) possessed almost no H1 histone kinase activity; however, activity was detected as early as 10 hr after cell activation when a protein (p60(Rb)) derived from the retinoblastoma susceptibility gene product was used as substrate. Cells were synchronized about the G1/S and G2/M borders by aphidicolin and nocodazole. Cells arrested prior to S-phase contained high levels of active p33(cdk2),nd essentially no active p34 despite the fact that large amounts of both proteins were present. Cells arrested by nocodazole had high levels of active p34(cdc2) and greatly reduced levels of p33(cdk2) kinase activity. The results suggest that the major role for the p34(cdc2) kinase is at mitosis, whereas that for p33(cdk2) is in late G1 and/or S phase. The p33(cdk4) protein, present in aphidicolin-blocked cells, was nearly absent from cells arrested at the G2/M border; however, kinase activity was low in cells blocked at both points, suggesting that the major role for p34(cdk4) may be in G1 phase. (C) 1995 Wiley-Liss, Inc.