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首页> 外文期刊>Molecular and Cellular Biology >Induced Expression of p16INK4a Inhibits Both CDK4- and CDK2-Associated Kinase Activity by Reassortment of Cyclin-CDK-Inhibitor Complexes
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Induced Expression of p16INK4a Inhibits Both CDK4- and CDK2-Associated Kinase Activity by Reassortment of Cyclin-CDK-Inhibitor Complexes

机译：诱导的p16INK4a的表达通过细胞周期蛋白-CDK-抑制剂复合物的重组抑制CDK4-和CDK2-相关的激酶活性

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To investigate the mode of action of the p16INK4a tumor suppressor protein, we have established U2-OS cells in which the expression of p16INK4a can be regulated by addition or removal of isopropyl-β-d-thiogalactopyranoside. As expected, induction of p16INK4a results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16INK4a also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27KIP1. Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16INK4a, this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16INK4a. Sequestration of CDK4 by p16INK4a allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16INK4a, p27KIP1appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27KIP1 and p21CIP1. Significantly, p16INK4a itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.
机译：为了研究p16 INK4a 肿瘤抑制蛋白的作用方式，我们建立了U2-OS细胞，其中p16 INK4a表达 em> 可以通过添加或去除异丙基-β-d-硫代半乳糖吡喃糖苷来调节。不出所料，p16 INK4a 的诱导通过抑制细胞周期蛋白-磷酸化成视网膜细胞瘤蛋白（pRb）导致G 1 细胞周期停滞。依赖性激酶CDK4和CDK6。但是，p16 INK4a 的诱导也引起CDK2活性的明显抑制。就细胞周期蛋白E-CDK2而言，这是由于细胞周期蛋白，CDK和CDK抑制剂复合物（尤其是涉及p27 KIP1 的复合物）的重新组合引起的。细胞裂解物的大小分级显示，大部分CDK4参与约200 kDa的活性激酶复合物。诱导p16 INK4a 后，该复合物部分解离，大部分CDK4存在于低分子量组分中，与p16二元复合物的形成相一致 INK4a 。 p16 INK4a 隔离CDK4可使细胞周期蛋白D1与CDK2的结合越来越紧密，而又不影响其与CIP / KIP抑制剂的相互作用。因此，在诱导p16 INK4a 时，p27 KIP1 似乎将其效忠从CDK4切换到CDK2，并且伴随的组分重配导致p27 KIP1 和p21 CIP1 抑制细胞周期蛋白E-CDK2。值得注意的是，p16 INK4a 本身似乎并不形成高阶复合物，绝大多数仍然保持自由或与CDK4和CDK6形成二元缔合。

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