Constitutively active protein phosphatase 1 alpha causes Rb-dependent G1 arrest in human cancer cells
Constitutively active protein phosphatase 1 alpha causes Rb-dependent G1 arrest in human cancer cells
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DOI：
10.1016/s0960-9822(06)00185-0
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发表时间：
1997-06-01
期刊：
影响因子：
9.2
通讯作者：
Liu, CWY
中科院分区：
文献类型：
--
作者：
Berndt, N;Dohadwala, M;Liu, CWY
背景资料：视网膜母细胞瘤蛋白（Rb）需要被细胞周期蛋白依赖性激酶（CDK）磷酸化，然后哺乳动物细胞才能进入细胞周期的S期。由于蛋白磷酸酶1（PP 1）激活Rb，并且其本身是体外CDK抑制磷酸化的靶点，我们询问PP 1对细胞周期进程的任何影响是否依赖于其磷酸化并通过Rb介导。利用重组蛋白电转移到Rb阳性和Rb阴性细胞中，我们比较了野生型PP 1催化亚基PP 1 α，以及该亚基的组成型活性突变体（PP 1 α T320 A）对G1期进展、增殖速率和细胞活力的影响。在处理的细胞中，PP 1 α水平升高6-16倍，并保持稳定至少48小时。在Rb阳性细胞中，PP 1 α T320 A，而不是PP 1 α，导致细胞周期停滞在G1晚期，这与Rb磷酸化的缺乏有关。在Rb阴性细胞中，无论是野生型还是突变型磷酸酶引起细胞周期进程的任何变化。然而，在Rb阳性和Rb阴性细胞中观察到细胞死亡增加，在引入过量的PP 1 α后。野生型和突变型PP 1 α的作用之间的差异表明，PP 1 α具有阻止G1期细胞生长的潜力，除非它被Thr 320的周期性磷酸化灭活，推测是通过调节通过G1-S细胞周期转换的CDK。总之，在两种细胞类型中的作用表明，PP 1 α需要功能性Rb来诱导生长停滞，并且可能另一个PP 1 α库诱导细胞死亡。这将PP 1确定为治疗性抗增殖策略的潜在靶点。
Background: The retinoblastoma protein (Rb) needs to be phosphorylated by cyclin-dependent kinases (CDKs) before mammalian cells can enter the S phase of the cell cycle. As protein phosphatase 1 (PP1) activates Rb and is itself a target for inhibitory phosphorylation by CDKs in vitro, we asked whether any effects of PP1 on cell cycle progression depend on its phosphorylation and are mediated through Rb.Results: Using electrotransfer of recombinant protein into Rb-positive and Rb-negative cells, we have compared the effects of a wild-type PP1 catalytic subunit, PP1 alpha, and a constitutively active mutant of this subunit (PP1 alpha T320A) on G1 progression, proliferation rates, and cell viability. In treated cells, PP1 alpha levels were elevated 6-16-fold and remained stable for at least 48 hours. In Rb-positive cells, PP1 alpha T320A, but not PP1 alpha, caused cell cycle arrest in late G1, which was associated with a lack of Rb phosphorylation. In Rb-negative cells, neither wild-type nor mutant phosphatase caused any change in cell cycle progression. Increased cell death was observed in both Rb-positive and Rb-negative cells, however, upon introduction of excess PP1 alpha.Conclusions: The difference between the effects of wild-type and mutant forms of PP1 alpha suggests that PP1 alpha has the potential to arrest cell growth in G1 unless it is inactivated by periodic phosphorylation at Thr320, presumably by CDKs that regulate passage through the G1-S cell cycle transition. Together, the effects in both cell types suggest that PP1 alpha requires functional Rb to induce growth arrest, and that possibly another pool of PP1 alpha induces cell death. This identifies PP1 as a potential target for therapeutic anti-proliferative strategies.