Garriga Judit, Jayaraman Arun L, Limón Ana, Jayadeva Girish, Sotillo Elena, Truongcao May, Patsialou Antonia, Wadzinski Brian E, Graña Xavier
Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Cell Cycle. 2004 Oct;3(10):1320-30. doi: 10.4161/cc.3.10.1183. Epub 2004 Oct 15.
It is thought that G(1) cyclin/CDK mediated phosphorylation of pocket proteins from mid G(1) to mitosis is reversed via dephosphorylation in mitosis. We examined the mechanisms involved in the unexpectedly rapid dephosphorylation of the pocket proteins induced via inhibition of cellular protein synthesis by cycloheximide (CHX) as well as direct inhibition of CDKs by flavopiridol. CHX and flavopiridol-induced dephosphorylation of pocket proteins is attributable to inactivation of D-type cyclin/CDKs and G(1)/S CDKs, respectively, which unmasks a phosphatase activity that targets the three pocket proteins apparently throughout the cell cycle. Treatment of cells with phosphatase inhibitors at concentrations selective for PP2A inhibition prevents CHX and flavopiridol-mediated dephosphorylation of pocket proteins in vivo. Also, ectopic expression of SV40 small t antigen, which inhibits PP2A via disruption of trimeric PP2A holoenzymes, delays CHX-induced pocket protein dephosphorylation. Moreover, dephosphorylation of p130 and p107 in cell extracts is inhibited by concentrations of okadaic acid known to inhibit PP2A, but not PP1. Finally, the PP2A catalytic subunit (PP2A/C) specifically interacts with both p130 and p107 in quiescent cells as well as cells progressing throughout the cell cycle. Together, these results demonstrate that the overall phosphorylation state of pocket proteins is determined, at least in part, by a dynamic equilibrium between CDKs and PP2A, or a closely related PP2A-like enzyme. These findings have important implications, as cell cycle or checkpoint-dependent inhibition of CDK activities counteracted by an active PP2A should have imminent effects on the phosphorylation state and activities of pocket proteins.
人们认为，从G1中期到有丝分裂期间，G1细胞周期蛋白/周期蛋白依赖性激酶（CDK）介导的口袋蛋白磷酸化在有丝分裂过程中通过去磷酸化而逆转。我们研究了通过环己酰亚胺（CHX）抑制细胞蛋白质合成以及通过黄酮哌啶醇直接抑制CDK所诱导的口袋蛋白意外快速去磷酸化所涉及的机制。CHX和黄酮哌啶醇诱导的口袋蛋白去磷酸化分别归因于D型细胞周期蛋白/CDK和G1/S CDK的失活，这揭示了一种明显在整个细胞周期靶向三种口袋蛋白的磷酸酶活性。用对PP2A抑制具有选择性的浓度的磷酸酶抑制剂处理细胞可在体内防止CHX和黄酮哌啶醇介导的口袋蛋白去磷酸化。此外，通过破坏三聚体PP2A全酶来抑制PP2A的SV40小t抗原的异位表达延迟了CHX诱导的口袋蛋白去磷酸化。此外，已知抑制PP2A而不抑制PP1的冈田酸浓度可抑制细胞提取物中p130和p107的去磷酸化。最后，PP2A催化亚基（PP2A/C）在静止细胞以及整个细胞周期进展的细胞中与p130和p107特异性相互作用。总之，这些结果表明，口袋蛋白的整体磷酸化状态至少部分地由CDK和PP2A或密切相关的PP2A样酶之间的动态平衡决定。这些发现具有重要意义，因为细胞周期或检查点依赖性的CDK活性抑制被活跃的PP2A抵消后，应该会对口袋蛋白的磷酸化状态和活性产生即时影响。
