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首页> 外文期刊>Molecular and Cellular Biology >A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.
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A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

机译：显性阴性的细胞周期蛋白D1突变体可防止细胞周期蛋白依赖性激酶4（CDK4）的核输入及其CDK激活激酶的磷酸化作用。

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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.
机译：细胞周期蛋白含有两个特征性细胞周期蛋白折叠，每个折叠由五个α-螺旋束组成，它们通过短接头肽相互连接。第一个重复与组装的全酶复合物中的细胞周期蛋白依赖性激酶（CDK）亚基直接接触，而第二个则不直接与CDK界面有关。尽管预计小鼠细胞周期蛋白D1中的苏氨酸156位于将两个细胞周期蛋白折叠折叠并埋藏在细胞周期蛋白亚基内的接头肽的羧基末端，但该残基突变为丙氨酸对衍生细胞周期蛋白D1的行为具有深远影响-CDK4复合物。与突变型细胞周期蛋白D1（T156A或T156E但不是T156S）形成复合体的CDK4在体外没有被重组CDK激活激酶（CAK）磷酸化，无法在体内经历激活的T环磷酸化作用，并且仍然没有催化活性并且无法将其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表达时，细胞周期蛋白D1（T156A）在细胞质中与CDK4组装在一起，但不会导入细胞核中。周期蛋白D1-CDK4复合物的核转运不需要CAK磷酸化，因为有效导入了含有野生型细胞周期蛋白D1和缺少CAK磷酸化位点的CDK4（T172A）突变体的复合物。相反，CDK抑制剂p21Cip1与突变细胞周期蛋白D1（T156A）-CDK4复合物的强制过度表达增强了它们的核定位。这些结果表明，细胞周期蛋白D1（T156A或T156E）与CDK4形成了流产复合物，从而阻止了CAK及其核定位所需的其他细胞因子的识别。这些特性使异位过表达的细胞周期蛋白D1（T156A）或对泛素化有抵抗力的更稳定的T156A / T286A双突变体能够与哺乳动物细胞中的内源性细胞周期蛋白D1竞争，从而将CDK4转移到细胞质，催化失活的复合物中并显着抑制其能力转染的NIH 3T3成纤维细胞进入S期。

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