Regazzi R, Li G, Ullrich S, Jaggi C, Wollheim C B
Institut de Biochimie Clinique, Centre Médical Universitaire, Geneva, Switzerland.
J Biol Chem. 1989 Jun 15;264(17):9939-44.
Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.
用电透化的RINm5F细胞来评估激活蛋白激酶C（PKC）和胰岛素分泌所需的因素。PKC可通过佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）或通过响应不可水解的鸟嘌呤核苷酸类似物鸟苷5'-O-（硫代三磷酸）（GTPγS）产生内源性二酰基甘油来激活。如先前所示，PMA和GTPγS均可引发不依赖Ca2 +的胰岛素分泌。这种效应被鸟苷-5'-基亚氨基二磷酸（Gpp(NH)p）模拟，但未被γ-磷酸基团中仅带有一个负电荷的鸟苷5'-O-（3 - 氟三磷酸）和鸟苷5'-O-（3 - 苯基三磷酸）模拟。PMA的作用由PKC介导，因为该试剂导致特定蛋白质底物的磷酸化以及该酶与细胞膜的结合。这种转位与所采用的Ca2 +浓度无关。相反，GTPγS仅在10^(-6)和10^(-5) M Ca2 +时促进PKC与膜的结合，并且在没有Ca2 +的情况下未能显著改变蛋白质磷酸化。刺激胰岛素释放的Gpp(NH)p以及其他两种GTP类似物均未增加与膜结合的PKC比例。为了验证GTPγS对PKC的Ca2 +依赖性作用是否归因于磷脂酶C的激活，我们测量了二酰基甘油的生成。在10^(-7)至10^(-5) M的Ca2 +浓度下，GTPγS确实刺激了渗漏细胞中二酰基甘油的产生约50％，在没有Ca2 +的情况下这种效应几乎消失。因此，在静息完整细胞中发现的10^(-7) M Ca2 +浓度下，生成的二酰基甘油不足以导致PKC插入膜中，这表明升高的Ca2 +和二酰基甘油对于转位的发生都是必需的。结论是，虽然PMA激活PKC引发不依赖Ca2 +的胰岛素分泌，但在没有Ca2 +的情况下，该激酶似乎不介导GTP类似物的刺激作用。
