Gopalakrishna R, Barsky S H, Thomas T P, Anderson W B
J Biol Chem. 1986 Dec 15;261(35):16438-45.
One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.
肿瘤促进剂佛波酯处理细胞的早期事件之一是蛋白激酶C与质膜紧密结合。为了更好地理解调节这一过程的因素，使用匀浆、分离的膜和纯化的酶研究了佛波酯诱导的蛋白激酶C的膜结合。在Ca2+存在的情况下，向壁层卵黄囊细胞和NIH 3T3细胞的匀浆中添加12 - O - 十四酰佛波醇13 - 乙酸酯（TPA）会导致蛋白激酶C与质膜结合，随后该结合与Ca2+无关而持续存在于膜上。尽管在无Ca2+时蛋白激酶C被TPA激活，在有Ca2+时被二油精激活，但在这些各自的条件下将这两种试剂添加到匀浆中时，它们对蛋白激酶C的膜结合均无影响。然而，在这些条件下，如果使用纯化的蛋白激酶C与分离的膜，则会发现蛋白激酶C的结合相对较弱。使用纯化的蛋白激酶C和洗涤过的膜进行的结合研究表明，TPA - 激酶复合物与膜的结合需要磷脂，并且在壁层卵黄囊细胞膜蛋白为0.1单位（24 ng蛋白激酶C）/mg时达到饱和。在有和无Ca2+的培养基中对细胞进行佛波酯处理表明，即使在完整细胞中，TPA诱导的膜相关蛋白激酶C的增加也受Ca2+水平调节。TPA稳定的蛋白激酶C的膜结合在几个方面不同于先前报道的Ca2+诱导的可逆结合。TPA稳定的蛋白激酶C与分离膜的结合是温度依赖性的，在富含质膜的部分中相对较高，在生理水平的蛋白激酶C时可饱和，需要膜蛋白和磷脂两者的存在，并且进一步需要添加磷脂微团。相比之下，Ca2+诱导的可逆结合更快，不受温度明显影响，对特定亚细胞部分无选择性，在生理量的蛋白激酶C时不饱和，表现出对胰蛋白酶不敏感的膜结合位点，并且需要膜磷脂但不需要添加的磷脂微团。
