Kojima I, Mogami H, Shibata H, Ogata E
Cell Biology Research Unit, Gunma University, Maebashi, Japan.
J Biol Chem. 1993 May 15;268(14):10003-6.
We previously reported that insulin-like growth factor-I (IGF-I) stimulated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and production of 1,2-diacylglycerol in IGF-responsive "primed competent" Balb/c 3T3 cells (Kojima, I., Kitaoka, M., and Ogata, E. (1990) J. Biol. Chem. 265, 16846-16850). The present study was conducted to determine a role of protein kinase C (PKC) in the progression activity of IGF-I. To monitor the activity of PKC in intact cells, we measured phosphorylation of a synthetic KRTLRR peptide, a substrate of PKC, immediately after the permeabilization of the cells with digitonin. When 1 nM IGF-I was added to primed competent cells, KRTLRR peptide phosphorylation was augmented. IGF-I induced more than 2-fold increase in KRTLRR peptide phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibitor of PKC. The phosphorylation remained elevated for up to 6 h. To assess the role of PKC in cell cycle progression, IGF-I-induced nuclear labeling was measured in the presence of Ro31-8220. Ro31-8220 reduced the rate of entrance into S phase when added in the first quarter of the G1 phase, but did not affect cell cycle progression when added at the second quarter or later. In contrast, reduction of extracellular calcium completely blocked cell cycle progression when done in the first, second, and third quarter but had no effect in the last quarter. These results indicate that IGF-I persistently activates PKC in primed competent cells, but the activation is required only for the initiation of progression. We conclude that IGF-I promotes cell cycle progression by calcium-dependent mechanisms that are largely independent of PKC.
我们之前报道过，胰岛素样生长因子-I（IGF-I）可刺激钙离子内流（小岛一、松永浩、黑川健、绪方英、西本一（1988年）《生物化学杂志》263卷，第16561 - 16567页）以及在对IGF有反应的“预处理有能力的”Balb/c 3T3细胞中1,2 - 二酰基甘油的产生（小岛一、北冈正、绪方英（1990年）《生物化学杂志》265卷，第16846 - 16850页）。本研究旨在确定蛋白激酶C（PKC）在IGF-I的促细胞增殖活性中的作用。为监测完整细胞中PKC的活性，在用洋地黄皂苷使细胞通透后，我们立即测量了PKC的底物合成KRTLRR肽的磷酸化情况。当向预处理有能力的细胞中添加1 nM IGF-I时，KRTLRR肽的磷酸化增强。IGF-I使KRTLRR肽的磷酸化增加了2倍以上，这种增加被PKC的假底物PKC19 - 36（可阻断激酶活性）和PKC抑制剂Ro31 - 8220所阻断。磷酸化水平持续升高长达6小时。为评估PKC在细胞周期进程中的作用，在存在Ro31 - 8220的情况下测量了IGF-I诱导的核标记。当在G1期的第一季度添加Ro31 - 8220时，其降低了进入S期的速率，但在第二季度或更晚添加时不影响细胞周期进程。相反，在第一、第二和第三季度降低细胞外钙会完全阻断细胞周期进程，但在最后一个季度则没有影响。这些结果表明，IGF-I在预处理有能力的细胞中持续激活PKC，但这种激活仅在增殖起始时是必需的。我们得出结论，IGF-I通过在很大程度上独立于PKC的钙依赖性机制促进细胞周期进程。
