Suzuki-Takahashi I, Kitagawa M, Saijo M, Higashi H, Ogino H, Matsumoto H, Taya Y, Nishimura S, Okuyama A
Banyu Tsukuba Research Institute, Merck Research Laboratories, Okubo, Japan.
Oncogene. 1995 May 4;10(9):1691-8.
It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
据推测，视网膜母细胞瘤基因的产物（pRB）在被细胞周期蛋白依赖性激酶（cdk）磷酸化后会从E2F-pRB复合物中解离。然而，目前尚无直接证据表明纯化的cdk通过磷酸化作用调控E2F-pRB复合物的形成。因此，我们使用pRB以及纯化的细胞周期蛋白A-cdk2、细胞周期蛋白E-cdk2或细胞周期蛋白D1-cdk4，研究了磷酸化作用对该复合物形成的调控。将纯化的pRB与从pRB缺陷细胞制备的核提取物一起孵育，然后进行凝胶迁移率变动分析。我们证实，未磷酸化的pRB与各种类型的E2F结合，但被细胞周期蛋白A-cdk2磷酸化后的pRB则不能。我们发现，细胞周期蛋白A-cdk2的磷酸化作用导致E2F-pRB复合物被破坏，E2F游离形式的水平增加。我们还发现，细胞周期蛋白A-cdk2的磷酸化作用不仅会破坏E2F-pRB复合物，还会破坏E2F-p107复合物。此外，细胞周期蛋白D1-cdk4和细胞周期蛋白E-cdk2以及细胞周期蛋白A-cdk2的磷酸化作用都会破坏E2F-pRB复合物。这些结果清楚地表明，cdk对pRB和p107的磷酸化作用调控了E2F与pRB或p107之间复合物的形成。
