Calbó Joaquim, Parreño Matilde, Sotillo Elena, Yong Thomas, Mazo Adela, Garriga Judit, Grana Xavier
Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2002 Dec 27;277(52):50263-74. doi: 10.1074/jbc.M209181200. Epub 2002 Oct 24.
Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.
有丝分裂原刺激导致G(1)细胞周期蛋白依赖性激酶（CDK）活化，这些激酶使口袋蛋白磷酸化并触发细胞周期通过G(0)/G(1)和G(1)/S转换。然而，G(1)细胞周期蛋白-CDK复合物在口袋蛋白的协同调节及其与E2F家族成员相互作用中的个体作用尚未完全了解。在此，我们报告，细胞周期蛋白D1-CDK和细胞周期蛋白E-CDK复合物单独或协同作用可诱导内源性口袋蛋白发生不同且协同的磷酸化，这在口袋蛋白与E2F4相互作用的调节以及p107和E2F1（这两个均受E2F调节的基因）的表达方面也有不同的后果。这两种蛋白的上调以及p130和pRB从E2F4复合物中的释放不仅允许E2F1复合物与pRB形成，还能与p130和p107形成，以及形成p107-E2F4复合物。这些复合物的形成发生在活性细胞周期蛋白D1-CDK和细胞周期蛋白E-CDK复合物存在的情况下，表明虽然磷酸化在消除某些口袋蛋白/E2F相互作用中起作用，但这些相同的活性在表达内源性水平口袋蛋白和E2F蛋白的细胞环境中诱导形成其他复合物。值得注意的是，不与E2F4相互作用的磷酸化p130“形式3”很容易与E2F1相互作用。我们的数据还表明，细胞周期蛋白的异位过表达足以在人T98G和大鼠-1细胞中诱导不依赖有丝分裂原的生长，尽管细胞周期蛋白D1的作用需要细胞周期蛋白E-CDK2活性的下游激活。有趣的是，在T98G细胞中，细胞周期蛋白D1比细胞周期蛋白E更有效地诱导细胞周期进程。这表明细胞周期蛋白D1独立于细胞周期蛋白E激活确保细胞周期及时进程的途径。
