Douglas D N, Fink H S, Rosé S D, Ridgway N D, Cook H W, Byers D M
Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Biochim Biophys Acta. 1997 Apr 24;1356(2):121-30. doi: 10.1016/s0167-4889(96)00164-4.
MARCKS (myristoylated alanine-rich C-kinase substrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC alpha to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC alpha in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.
MARCKS（肉豆蔻酰化富含丙氨酸的C激酶底物）已知在一个中央碱性结构域与钙调蛋白、肌动蛋白丝和阴离子磷脂相互作用，该结构域也是蛋白激酶C（PKC）磷酸化的位点。在本研究中，细胞松弛素D（CD）和钙调蛋白拮抗剂被用于研究F-肌动蛋白和钙调蛋白对C6胶质瘤细胞中MARCKS膜相互作用的影响。用CD处理1小时会破坏F-肌动蛋白丝，增加膜结合的免疫反应性MARCKS（从总量的51%增加到62%），但显著增强了响应佛波酯4β-12-O-十四烷酰佛波醇13-乙酸酯而转移到胞质部分的MARCKS量。相比之下，CD处理对佛波酯刺激的MARCKS磷酸化或PKCα向膜部分的转位没有影响。星形孢菌素也以不依赖PKC的方式增加了MARCKS与膜的结合，因为未观察到MARCKS磷酸化的变化，且双吲哚马来酰亚胺（一种更特异的PKC抑制剂）未改变MARCKS的分布。在CD预处理和未处理的细胞中，星形孢菌素均抑制佛波酯诱导的MARCKS转位，但不抑制PKCα的转位。钙调蛋白拮抗剂（三氟拉嗪、氯米帕明）对MARCKS的细胞分布或磷酸化影响很小，但与佛波酯协同作用，使MARCKS从膜上转位，而其磷酸化没有进一步增加。我们得出结论，响应激活的PKC时，MARCKS的磷酸化和转位不需要细胞骨架完整性，但与F-肌动蛋白和钙调蛋白的相互作用可能独立调节PKC调控的MARCKS在细胞膜上的定位和功能。
