Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression
Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression
复制标题
DOI：
10.1074/jbc.m007992200
复制
发表时间：
2001-04-06
影响因子：
4.8
通讯作者：
Hatakeyama, M
中科院分区：
文献类型：
--
作者：
Ashizawa, S;Nishizawa, H;Hatakeyama, M
视网膜母细胞瘤蛋白pRb的活性受G(1)细胞周期蛋白相关的细胞周期蛋白依赖性蛋白激酶(CDKs)介导的磷酸化调控。由于与pRb相关的口袋蛋白p107和p130具有与pRb相同的一般结构和生物学功能，因此它们的活性也被认为是由磷酸化调节的。在这项工作中，我们通过用非磷酸化的丙氨酸残基取代潜在的细胞周期蛋白CDK磷酸化位点来产生耐磷酸化的p107和p130分子。这些磷酸化抗性突变体保留了结合E2F和细胞周期蛋白的能力。在p16(INK4a)缺陷的U2-OS骨肉瘤细胞中，细胞周期蛋白D-CDK4/6调控失调，而野生型p107或p130不能抑制细胞增殖。此外，当在pRb缺陷的SAOS-8骨肉瘤细胞中异位表达时，野生型和抗磷酸化的pRb家族蛋白能够诱导大的扁平细胞。野生型蛋白的平坦化诱导活性，但不是磷酸化抗性突变体的活性：被共表达周期蛋白E所消除。我们的结果表明，周期蛋白D或周期蛋白E相关蛋白的升高导致了pRb家族蛋白的全身性失活，并表明与Rb基因的遗传失活相比，pRb激酶的失调在更广泛的细胞类型中引起了细胞周期的异常。
The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-8 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants; was:abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.