Schulman B A, Lindstrom D L, Harlow E
Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129, USA.
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10453-8. doi: 10.1073/pnas.95.18.10453.
An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is approximately 35A away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A-substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A's ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein-protein interaction distant from the active site is critical for phosphorylation by cdks.
细胞周期领域的一个重要问题是细胞周期蛋白依赖性激酶（cdks）如何靶向其底物。我们研究了细胞周期蛋白A表面保守疏水区域在细胞周期蛋白A-cdk2识别底物中的作用。这个疏水区域距离cdk2的活性位点约35埃，包含许多哺乳动物细胞周期蛋白中保守的MRAIL序列。在细胞周期蛋白A-cdk2-p27的X射线结构中，这个疏水区域与p27中的RNLFG序列接触，该序列在许多哺乳动物cdk的底物和抑制剂中都很常见。我们发现，细胞周期蛋白A上这个疏水区域的突变消除了与含有RXL基序的蛋白质的结合，而不影响与cdk2的结合。这个对接位点对于细胞周期蛋白A-cdk2对含有RXL基序的底物的磷酸化至关重要，但对组蛋白H1的磷酸化不重要。细胞周期蛋白对底物结合的受损是RXL底物磷酸化缺陷的原因，因为通过以异源方式恢复细胞周期蛋白A与底物的相互作用可以挽救磷酸化。此外，保守的疏水区域对细胞周期蛋白A在细胞中的功能很重要，有助于细胞周期蛋白A驱动细胞脱离细胞周期的G1期的能力。因此，我们定义了一种细胞周期蛋白可以将底物招募到cdks的机制，我们的结果支持这样一种观点，即由远离活性位点的蛋白质-蛋白质相互作用提供的高局部底物浓度对于cdks的磷酸化至关重要。
