Eckberg W R, Szuts E Z, Carroll A G
Department of Zoology, Howard University, Washington, D.C. 20059.
Dev Biol. 1987 Nov;124(1):57-64. doi: 10.1016/0012-1606(87)90459-3.
To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.
为了测试蛋白激酶C（C激酶）在调节鸟蛤卵母细胞生发泡破裂（GVBD）中可能发挥的作用，我们研究了佛波酯和C激酶拮抗剂对GVBD和蛋白质磷酸化的影响。将对这些试剂的反应与受精或细胞外钾离子增加所引发的反应进行比较。促肿瘤佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）是一种有效的C激酶激动剂，在20 nM时以半数最大刺激引发GVBD。相比之下，不刺激C激酶的佛波酯4α-佛波醇-12,13-十二烷酸酯不会引发GVBD。TPA在由过量钾离子诱导时加速了GVBD，但在受精引发该过程时不影响其时间进程。三种结构不同的C激酶拮抗剂（W-7、H-7和视黄醇）在受精或TPA诱导时均能阻断GVBD。当卵母细胞用[32P]正磷酸盐预孵育，然后通过受精、TPA或45 mM钾离子刺激使其发生GVBD时，蛋白质磷酸化大大增加，尤其是对于约45 kDa的一种或多种多肽。磷酸化在GVBD之前增加。视黄醇抑制活化卵中的磷酸化。在卵母细胞提取物中证实了C激酶活性。这些结果强烈表明，C激酶介导的蛋白质磷酸化参与了调节鸟蛤卵母细胞GVBD的途径。
