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B55alpha PP2A holoenzymes modulate the phosphorylation status of the pRB-related p107 and p130 proteins.

机译:B55alpha PP2A全酶调节与pRB相关的p107和p130蛋白的磷酸化状态。

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The retinoblastoma family of phosphoproteins consisting of the retinoblastoma protein (pRB) and the two structurally related proteins p130 and p107 play an important role in the negative regulation of cell cyle progression. Hypophosphorylated pocket proteins interact with the different members of the E2F family and repress the transcription of E2F-dependent genes and consequently suppress cell cycle progression through the G0/G1 transition and the restriction point in G1. Mitogenic stimulation results in sequential activation of cyclin/CDK complexes in mid to late G1, leading to subsequent hyperphosphorylation at multiple Ser/Thr sites of pocket proteins triggering dissociation of pocket protein/E2F complexes. This disruption leads to de-repression of many E2F dependent genes whose products are essential for cell cycle progression. The traditional view has been that pocket proteins continue to be hyperphosphorylated through the S and G2 phases and following cyclin/CDK inactivation during mitotic exit become dephosphorylated by action of PP1. However, our lab observed that upon treatment of asynchronously growing cells with the CDK inhibitor Flavopiridol or CHX, pocket proteins, are rapidly dephosphorylated correlating with the inactivation of G1/CDKs and down regulation of D-type cyclins, respectively. Pocket protein dephosphorylation was prevented by pre-treating these cells with phosphtase inhibitors at a concentration selective for PP2A, implicating PP2A or PP2A-like serine/threonine phosphatase in this process. The involvement of PP2A on pocket protein dephosphorylation was further strengthened by the observation that SV40 small t antigen (ST) delays/prevents p107 dephosphorylation. Moreover, a physical association between PP2A/C and p130/p107 was observed throughout the cell cycle that was not affected by CHX treatment, strongly suggesting that CHX-induced dephosphorylation is not the result of increased pocket protein targeting by PP2A, but rather that a dynamic equilibrium between CDKs and PP2A is shifted to dephosphorylation when CDK activity is compromised. This dynamic equilibrium operates throughout the cell cycle.;PP2A is a trimeric enzyme complex consisting of a catalytic C, a structural A and substrate specific B subunit. There are four families of regulatory B subunits designated B, B', B'' and B''', each with several members encoded by genes with multiple splice variants that mediate substrate specificity and subcellular localization. It has been reported recently that in excess of 200 functional distinct PP2A holoenzymes can assemble with distinct specificities. Therefore, to gain insight into the mechanisms that regulate the steady state phosphorylation of pocket proteins throughout the cell cycle, it was essential to identify the specific holoenzyme complexes involved. To this end, it was identified that a PP2A trimeric holoenzyme containing B55alpha specifically targets and dephosphorylates p107/p130 both in vitro and in mammalian cells. B55alpha associates directly with the spacer of p107 and this interaction seems to be indirectly enhanced by the C-terminus of p107. The decreased association of p107 with PP2A/C of the B55alpha/PP2A holoenzyme complex upon treatment with ST further confirmed the role of B55alpha in mediating p107-PP2A/C interaction. Our data also revealed an interaction between B55alpha and p130, but not pRb, which appears to prefer a PR70, suggesting selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes. In accordance with this, recombinant purified B55alpha dephosphorylates p107 in vitro. Limited ectopic expression of B55alpha but not other subunits, result in ST sensitive dephosphorylation of p107 and p130 in cells. Further shRNA mediated knockdown of B55alpha results in hyperphosphorylation of p107 and p130. This suggests that the cellular levels of B55alpha are critical in modulating the phosphorylation status of p107/p130 rather than just catalyzing the dephosphorylation of these proteins when the activity of CDKs is compromised. Since ST disrupts the B55alpha/PP2A holoenzyme complex by binding to the PP2A-A-C dimer and leads to hyperphosphorylation of pocket proteins it is conceivable that ST mediates its effects on cell proliferation at least in part, via inactivation of the PP2A holoenzymes that activates pocket proteins. Given the sensitivity of p107 phosphorylation to the cellular levels of B55alpha, future analyses should ascertain if deregulation of B55alpha leads to hyperphosphorylation of pocket proteins and abnormal cell cycle progression.
机译:由视网膜母细胞瘤蛋白(pRB)和两个与结构相关的蛋白p130和p107组成的视网膜母细胞瘤家族的磷蛋白在细胞周期进程的负调控中起重要作用。次磷酸化的口袋蛋白与E2F家族的不同成员相互作用,抑制E2F依赖性基因的转录,从而抑制细胞周期通过G0 / G1过渡和G1的限制点进行。促有丝分裂的刺激导致G1中晚期G1细胞周期蛋白/ CDK复合物的顺序活化,从而导致随后在多个口袋蛋白Ser / Thr位点发生超磷酸化,从而触发口袋蛋白/ E2F复合物的解离。这种破坏导致许多E2F依赖基因的去抑制,其产物对于细胞周期进程至关重要。传统观点认为,口袋蛋白在S和G2相中继续被过度磷酸化,在有丝分裂退出过程中,随着细胞周期蛋白/ CDK的失活,PP1的作用使其去磷酸化。但是,我们的实验室观察到,用CDK抑制剂Flavopiridol或CHX处理异步生长的细胞后,口袋蛋白迅速去磷酸化,分别与G1 / CDK的失活和D型细胞周期蛋白的下调相关。通过用对PP2A有选择性的浓度的磷酸酶抑制剂对这些细胞进行预处理来预防口袋蛋白的去磷酸化作用,在此过程中牵涉到PP2A或类似PP2A的丝氨酸/苏氨酸磷酸酶。通过观察到SV40小t抗原(ST)延迟/阻止了p107的去磷酸化作用,进一步加强了PP2A对口袋蛋白去磷酸化的参与。此外,在整个细胞周期中都观察到PP2A / C与p130 / p107之间存在物理联系,不受CHX处理的影响,这强烈表明CHX诱导的去磷酸化不是PP2A靶向口袋蛋白增加的结果,而是当CDK活性受到损害时,CDK和PP2A之间的动态平衡会转变为去磷酸化。这种动态平衡在整个细胞周期中都起作用。PP2A是三聚体酶复合物,由催化C,结构A和底物特异性B亚基组成。有四个调节性B亚基家族,分别命名为B,B',B''和B''',每个家族的成员均由具有多个介导底物特异性和亚细胞定位的剪接变体的基因编码。最近已经报道,超过200种功能不同的PP2A全酶可以以不同的特异性组装。因此,要深入了解调节整个细胞周期中袋装蛋白质稳态磷酸化的机制,鉴定涉及的特定全酶复合物至关重要。为此,已经确定了含有B55α的PP2A三聚全酶在体外和在哺乳动物细胞中特异性地靶向和去磷酸化p107 / p130。 B55alpha直接与p107的间隔子缔合,这种相互作用似乎被p107的C端间接增强。用ST处理后,p107与B55alpha / PP2A全酶复合物的PP2A / C的缔合减少,进一步证实了B55alpha在介导p107-PP2A / C相互作用中的作用。我们的数据还揭示了B55alpha和p130之间的相互作用,但不显示pRb,后者似乎更喜欢PR70,这表明口袋蛋白与不同的PP2A全酶之间的相互作用具有选择性。据此,重组纯化的B55alpha在体外使p107脱磷酸。 B55alpha的异位表达有限,但其他亚基的表达有限,导致p107和p130在细胞内发生ST敏感的去磷酸化。 shRNA介导的B55alpha的进一步敲低导致p107和p130的过度磷酸化。这表明B55alpha的细胞水平对于调节p107 / p130的磷酸化状态至关重要,而CDKs的活性受损时,它们仅催化这些蛋白的去磷酸化。由于ST通过与PP2A-AC二聚体结合破坏B55alpha / PP2A全酶复合物并导致口袋蛋白过度磷酸化,因此可以想象,ST至少部分地通过灭活激活口袋蛋白的PP2A全酶来介导其对细胞增殖的影响。 。鉴于p107磷酸化对B55alpha细胞水平的敏感性,未来的分析应确定B55alpha的失调是否导致口袋蛋白过度磷酸化和异常的细胞周期进程。


  • 作者

    Jayadeva, Girish.;

  • 作者单位

    Temple University.;

  • 授予单位 Temple University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 178 p.
  • 总页数 178
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词


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