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
Jj. Lucas et al., 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, Journal of cellular physiology, 165(2), 1995, pp. 406-416
Three major cyclin-dependent kinases, p34(cdc2), P33(cdk2) and p34(cdk
4) 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 cel
ls. Transcripts from the cdk4 and cdk2 genes were detectable as early
as 3 and 8 hr after stimulation, respectively, whereas cdc2 gene trans
cripts were not detectable until about 24 hr, shortly before S phase e
ntry. Immunoblot analysis showed that resting T cells contained little
p34(cdk4), no p34(cdc2), and a low level of p33(cdk2) protein. Increa
sed amounts of p34(cdk4), p33(cdk2) and p34(cdc2) proteins were seen a
t about 7, 10, and 30 hr after stimulation, respectively. Immunoprecip
itates of each of the kinases were assessed for histone H1 kinase acti
vity. Activity due to p33(cdk2) first became detectable in mid-G1 phas
e and increased dramatically after entry into S phase. Active p34(cdc2
) kinase was not detected until about 40 hr after stimulation, about 1
0 hr after the first appearance of the protein. Immunoprecipitates of
p34(cdk4) possessed almost no H1 histone kinase activity; however, act
ivity was detected as early as 10 hr after cell activation when a prot
ein (p60(Rb)) derived from the retinoblastoma susceptibility gene prod
uct 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 acti
ve p34 despite the fact that large amounts of both proteins were prese
nt. Cells arrested by nocodazole had high levels of active p34(cdc2) a
nd greatly reduced levels of p33(cdk2) kinase activity. The results su
ggest that the major role for the p34(cdc2) kinase is at mitosis, wher
eas that for p33(cdk2) is in late G1 and/or S phase. The p33(cdk4) pro
tein, present in aphidicolin-blocked cells, was nearly absent from cel
ls arrested at the G2/M border; however, kinase activity was low in ce
lls blocked at both points, suggesting that the major role for p34(cdk
4) may be in G1 phase. (C) 1995 Wiley-Liss, Inc.