Guida P, Zhu L
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Biochem Biophys Res Commun. 1999 May 19;258(3):596-604. doi: 10.1006/bbrc.1999.0656.
Stable enzyme-substrate interaction has been recognized as a major mechanism underlying the substrate preferences of cyclin-dependent kinases (Cdks). To learn the relationship between stability of physical association and efficiency of phosphorylation, we studied DP1 phosphorylation by cyclin A-Cdk2 in multiprotein complexes. When DP1 was connected to cyclin A-Cdk2 through E2F4 and p107, its phosphorylation was very inefficient, although its association with cyclin A-Cdk2 was stable. In contrast, DP1 was efficiently phosphorylated when weakly connected to cyclin A-Cdk2 via E2F1 or E2F4 with a fused cyclin A binding domain of E2F1. The transactivation activity of E2F4-DP1 heterodimers was reduced when DP1 was phosphorylated, while a phosphorylation deficient mutant of DP1 resisted this down-regulation. Phosphorylation and functional regulation of DP1 were not due to nuclear localization. Thus, stronger physical association between the kinase and the substrate does not necessarily lead to more efficient phosphorylation than weaker interaction does.
稳定的酶-底物相互作用已被认为是细胞周期蛋白依赖性激酶（Cdks）底物偏好的主要潜在机制。为了了解物理结合稳定性与磷酸化效率之间的关系，我们研究了多蛋白复合物中细胞周期蛋白A-Cdk2对DP1的磷酸化作用。当DP1通过E2F4和p107与细胞周期蛋白A-Cdk2相连时，尽管其与细胞周期蛋白A-Cdk2的结合稳定，但其磷酸化效率却非常低。相反，当DP1通过E2F1或带有E2F1融合细胞周期蛋白A结合域的E2F4与细胞周期蛋白A-Cdk2弱连接时，DP1能被有效磷酸化。当DP1被磷酸化时，E2F4-DP1异二聚体的反式激活活性降低，而DP1的磷酸化缺陷突变体则能抵抗这种下调。DP1的磷酸化和功能调节并非由于核定位。因此，激酶与底物之间更强的物理结合不一定比弱相互作用导致更有效的磷酸化。
