Diehl J A, Sherr C J
Howard Hughes Medical Institute, Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Mol Cell Biol. 1997 Dec;17(12):7362-74. doi: 10.1128/MCB.17.12.7362.
Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.
细胞周期蛋白包含两个特征性的细胞周期蛋白折叠结构，每个结构由五个α-螺旋束组成，它们通过一个短的连接肽相互连接。第一个重复序列在组装好的全酶复合物中与细胞周期蛋白依赖性激酶（CDK）亚基直接接触，而第二个重复序列并不直接参与CDK界面的形成。尽管小鼠细胞周期蛋白D1中的苏氨酸156预计位于分隔两个细胞周期蛋白折叠结构的连接肽的羧基末端，并埋在细胞周期蛋白亚基内，但将该残基突变为丙氨酸对衍生的细胞周期蛋白D1-CDK4复合物的行为有深远影响。与突变型细胞周期蛋白D1（T156A或T156E而非T156S）形成复合物的CDK4在体外不能被重组的CDK激活激酶（CAK）磷酸化，在体内不能进行激活T环的磷酸化，并且仍然没有催化活性，无法磷酸化视网膜母细胞瘤蛋白。此外，当它在哺乳动物细胞中异位过表达时，细胞周期蛋白D1（T156A）与CDK4在细胞质中组装，但不会被转运到细胞核中。细胞周期蛋白D1-CDK4复合物的核转运不需要CAK磷酸化，因为含有野生型细胞周期蛋白D1和缺乏CAK磷酸化位点的CDK4（T172A）突变体的复合物能有效转运。相反，CDK抑制剂p21Cip1与突变型细胞周期蛋白D1（T156A）-CDK4复合物一起强制过表达会增强它们的核定位。这些结果表明，细胞周期蛋白D1（T156A或T156E）与CDK4形成了失败的复合物，从而阻止了CAK和其他核定位所需细胞因子的识别。这些特性使异位过表达的细胞周期蛋白D1（T156A）或更稳定的对泛素化有抗性的T156A/T286A双突变体能在哺乳动物细胞中与内源性细胞周期蛋白D1竞争，从而将CDK4动员到细胞质中、无催化活性的复合物中，并显著抑制转染的NIH 3T3成纤维细胞进入S期的能力。
