Martinez A M, Afshar M, Martin F, Cavadore J C, Labbé J C, Dorée M
Centre de Recherches de Biochimie Macromoléculaire, CNRS ERS155 and INSERM U 249, Montpellier, France.
EMBO J. 1997 Jan 15;16(2):343-54. doi: 10.1093/emboj/16.2.343.
A cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown previously to catalyze T-loop phosphorylation of cdks in most eukaryotic cells. This enzyme exists in either of two forms: the major one contains cdk7, cyclin H and an assembly factor called MAT-1, whilst the minor one lacks MAT-1. Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T-loop that are phosphorylated in vivo. We have investigated the role of S170 and T176 phosphorylation in the assembly and activity of cyclin H-cdk7 dimers. In the absence of MAT-1, phosphorylation of the T-loop appears to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro. Phosphorylation of S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation. By mutational analysis, we demonstrate that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. Finally, we show that dual phosphorylation of S170 and T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one-third of its maximal value in vitro, even in the absence of cyclin H and MAT-1, and may thus provide further clues as to how cyclins activate cdk subunits.
先前已表明，在大多数真核细胞中，一种细胞周期蛋白依赖性激酶（cdk）激活激酶（CAK）可催化cdk的T环磷酸化。这种酶以两种形式存在：主要形式包含cdk7、细胞周期蛋白H和一种称为MAT-1的组装因子，而次要形式则缺乏MAT-1。Cdk7在cdk中较为特殊，因为其T环中在体内被磷酸化的不是一个而是两个残基（非洲爪蟾cdk7中的S170和T176）。我们研究了S170和T176磷酸化在细胞周期蛋白H-cdk7二聚体组装和活性中的作用。在没有MAT-1的情况下，T环的磷酸化似乎是cdk7结合细胞周期蛋白H所必需的。在体外，两个残基的磷酸化并不需要细胞周期蛋白H的结合。S170的磷酸化足以使cdk7以低亲和力结合细胞周期蛋白H，但高亲和力结合需要T176的磷酸化。通过突变分析，我们证明，除了在促进细胞周期蛋白H结合方面的作用外，S170磷酸化在CAK活性控制中也起直接作用。最后，我们表明，S170和T176的双重磷酸化，或将两个可磷酸化残基替换为天冬氨酸残基，即使在没有细胞周期蛋白H和MAT-1的情况下，也足以在体外产生高达其最大值三分之一的CAK活性，这可能因此为细胞周期蛋白如何激活cdk亚基提供进一步线索。
