Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1-to S-phase by control of cyclin E-CDK2 activity
Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1-to S-phase by control of cyclin E-CDK2 activity
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DOI：
10.1042/bj20080233
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发表时间：
2008-11-01
影响因子：
4.1
通讯作者：
Kelley, Grant G.
中科院分区：
文献类型：
--
作者：
Kaproth-Joslin, Katherine A.;Li, Xiangquan;Kelley, Grant G.
在本研究中，我们研究了PLC δ 1（磷脂酶C δ）在细胞增殖调节中的作用。我们证明，RNAi （RNA干扰）介导的内源性PLC δ 1的敲低，而不是PLC β 3或PLC epsilon，诱导了Rat-1和NTH 3T3成纤维细胞的增殖缺陷。细胞增殖的减少不是由于诱导细胞凋亡或衰老，而是与细胞凋亡有关。60%抑制[H-3]胸苷结合。用BrdU（溴脱氧尿苷）/碘化丙啶标记的FACS（荧光活化细胞分选）分析细胞周期，发现G(0)/G(1)期细胞积累，s期细胞相应减少。血清饥饿同步后对细胞周期的进一步检查显示G(1)期运动正常，但进入s期延迟。与这些发现一致，G、细胞周期蛋白（D2和D3）和CDK4（细胞周期蛋白依赖性激酶4）水平和相关的激酶活性没有受到影响。然而，负责G(1)到s期进展的细胞周期蛋白e相关CDK2活性被抑制。这种活性降低伴随着CDK2蛋白水平不变和细胞周期蛋白E和细胞周期蛋白E相关的CDK2水平矛盾地升高，表明细胞周期蛋白E-CDK2复合物受到抑制。这种抑制不是由于CDK2的刺激或抑制性磷酸化改变。然而，Cip/Kip家族CKI （CDK抑制剂）结合伙伴p27在PLC δ 1敲低的细胞中升高，并显示与CDK2的关联增加。本研究的结果表明，PLC δ 1通过调节细胞周期蛋白E-CDK2活性和p27水平，在细胞周期从G(1)期到s期的过程中发挥了新的关键作用。
In the present study, we examined the role of PLC delta 1 (phospholipase C delta) in the regulation Of Cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLC delta 1, but not PLC beta 3 or PLC epsilon, induces a proliferation defect in Rat-1 and NTH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [H-3]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G(0)/G(1)-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G(1)-phase but delayed entry into S-phase. Consistent with these findings, G, cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G(1)-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E-CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLC delta 1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLC delta 1 in cell-cycle progression from G(1)-to-S-phase through regulation of cyclin E-CDK2 activity and p27 levels.