Yee A, Wu L, Liu L, Kobayashi R, Xiong Y, Hall F L
Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles 90033, USA.
J Biol Chem. 1996 Jan 5;271(1):471-7. doi: 10.1074/jbc.271.1.471.
The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.
细胞周期蛋白依赖性蛋白激酶（Cdks）的激活依赖于位点特异性磷酸化和去磷酸化反应，以及正性和负性调节亚基。人Cdk激活蛋白激酶（Cak1）本身是一种与细胞周期蛋白H相关的Cdc2相关细胞周期蛋白依赖性蛋白激酶。本研究利用特异性抗Cak1抗体和免疫亲和色谱法来鉴定其他与Cak1相关的蛋白和潜在的靶底物。对代谢标记的人骨肉瘤细胞进行免疫沉淀，发现了许多与Cak1相关的蛋白，包括p95、p37（细胞周期蛋白H）和一种在此进一步鉴定的35 kDa蛋白。有限蛋白酶解后进行的微序列分析揭示了与人和酵母细胞周期蛋白相似但不完全相同的肽片段，从而将p35鉴定为一种细胞周期蛋白样调节亚基。人p35与Mcs2的序列相似性最高，Mcs2是一种对细胞周期进程至关重要的酵母细胞周期蛋白。在非变性条件下进行的免疫亲和色谱法能够从细胞裂解物中分离出具有酶活性的Cak1，从而能够研究激酶的自磷酸化和比较底物利用情况。免疫亲和纯化的Cak1能磷酸化单体Cdc2和Cdk2，但不能磷酸化Cdk4；在重组细胞周期蛋白A存在的情况下，Cdc2和Cdk2的磷酸化均增加。这些研究表明，Cak1催化亚基与Cdc2和Cdk2一样，与多个调节伙伴相关联，提示亚基组成可能是这种多功能酶的一个重要决定因素。
