New functional activities for the p21 family of CDK inhibitors
New functional activities for the p21 family of CDK inhibitors
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DOI：
10.1101/gad.11.7.847
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发表时间：
1997-04-01
影响因子：
10.5
通讯作者：
Harlow, E
中科院分区：
文献类型：
--
作者：
LaBaer, J;Garrett, MD;Harlow, E
cdk 4与D-型细胞周期蛋白结合形成功能性激酶复合物的效率相对较低。这导致有人建议，组装可能是一个受管制的步骤。在这份报告中，我们证明了CDK抑制剂p21（CIP），p27（KIP）和p57（KIP 2）都促进了CDK 4与D型细胞周期蛋白的结合。这种效应是特异性的，并且不会发生在其他cdk抑制剂或cdk结合蛋白上。在体内和体外，组装的cdk 4/细胞周期蛋白D复合物的丰度直接随着抑制剂水平的增加而增加。促进装配不是归因于一个简单的细胞周期阻滞，需要的cdk和细胞周期蛋白结合域的功能。动力学研究表明，p21和p27分别导致K-a增加35倍和80倍，主要是因为K-off减少。在低浓度下，p21促进活性激酶复合物的组装，而在较高浓度下，它抑制活性。此外，免疫耗竭实验表明，大多数活性cdk 4相关激酶活性也与p21相关。为了在自然环境中证实这些结果，我们研究了从G（0）停滞释放后乳腺上皮细胞中内源性复合物的组装。在协议与我们的其他数据，细胞周期蛋白D1和p21结合伴随cdk 4在体内组装的cdk 4/细胞周期蛋白D1复合物。这种复杂的装配发生在平行的细胞周期蛋白D1相关激酶活性的增加。免疫耗竭实验表明，大多数细胞周期蛋白D1相关激酶活性也与p21相关。最后，我们发现所有三种CIP/KIP抑制剂都将cdk 4和cyclin D1靶向细胞核。我们认为，除了它们作为抑制剂的作用，p21蛋白家族，最初被确定为抑制剂，也可能有作为适配器蛋白组装和程序激酶复合物的特定功能的作用。
The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K-a, respectively, mostly because of a decrease in K-off. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest. In agreement with our other data, cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.