Lucas J J, Szepesi A, Domenico J, Tordai A, Terada N, Gelfand E W
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
J Cell Physiol. 1995 Nov;165(2):406-16. doi: 10.1002/jcp.1041650222.
Three major cyclin-dependent kinases, p34cdc2, p33cdk2, and p34cdk4 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 form 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 p34cdk4, no p34cdc2, and a low level of p33cdk2 protein. Increased amounts of p34cdk4, p33cdk2, and p34cdc2 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 p33cdk2 first became detectable in mid-G1 phase and increased dramatically after entry into S phase. Active p34cdc2 kinase was not detected until about 40 hr after stimulation, about 10 hr after the first appearance of the protein. Immunoprecipitates of p34cdk4 possessed almost no H1 histone kinase activity; however, activity was detected as early as 10 hr after cell activation when a protein (p60Rb) 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 p33cdk2 and essentially no active p34cdc2, despite the fact that large amounts of both proteins were present. Cells arrested by nocodazole had high levels of active p34cdc2 and greatly reduced levels of p33cdk2 kinase activity. The results suggest that the major role for the p34cdc2 kinase is at mitosis, whereas that for p33cdk2 is in late G1 and/or S phase. The p34cdk4 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 p34cdk4 may be in G1 phase.
在体外刺激进入细胞周期的正常人T细胞中检测了三种主要的细胞周期蛋白依赖性激酶，即p34cdc2、p33cdk2和p34cdk4。三种基因在静止T细胞中均不表达。cdk4和cdk2基因的转录本分别在刺激后3小时和8小时即可检测到，而cdc2基因转录本直到约24小时，即进入S期前不久才检测到。免疫印迹分析表明，静止T细胞中p34cdk4含量很少，没有p34cdc2，p33cdk2蛋白水平较低。分别在刺激后约7小时、10小时和30小时观察到p34cdk4、p33cdk2和p34cdc2蛋白含量增加。对每种激酶的免疫沉淀物进行组蛋白H1激酶活性评估。p33cdk2引起的活性最早在G1期中期可检测到，并在进入S期后显著增加。直到刺激后约40小时，即该蛋白首次出现后约10小时，才检测到活性p34cdc2激酶。p34cdk4的免疫沉淀物几乎没有H1组蛋白激酶活性；然而，当来自视网膜母细胞瘤易感基因产物的一种蛋白（p60Rb）用作底物时，在细胞活化后10小时就检测到了活性。用阿非科林和诺考达唑使细胞在G1/S和G2/M边界同步化。尽管两种蛋白都大量存在，但在S期之前停滞的细胞含有高水平的活性p33cdk2，基本上没有活性p34cdc2。被诺考达唑阻滞的细胞具有高水平的活性p34cdc2，而p33cdk2激酶活性水平大大降低。结果表明，p34cdc2激酶的主要作用在有丝分裂期，而p33cdk2的作用在G1晚期和/或S期。在阿非科林阻滞的细胞中存在的p34cdk4蛋白，在G2/M边界停滞的细胞中几乎不存在；然而，在这两个点阻滞的细胞中激酶活性都很低，这表明p34cdk4的主要作用可能在G1期。
