Elzagallaai A, Rosé S D, Trifaró J M
Secretory Process Research Program, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Blood. 2000 Feb 1;95(3):894-902.
Previous experiments suggest that actin disassembly, perhaps at a specific site, is required for platelet secretion. Platelet stimulation by phorbol 12-myristate 13-acetate (PMA) induced pleckstrin phosphorylation, platelet aggregation, and secretion. Inhibition of protein kinase C (PKC) is accompanied by inhibition of pleckstrin phosphorylation and serotonin secretion. Here, we demonstrate the presence of myristoylated alanine-rich C kinase substrate (MARCKS), another PKC substrate, in platelets and its phosphorylation during PMA stimulation. MARCKS is known to bind actin and to cross-link actin filaments; the latter is inhibited by PKC-induced MARCKS phosphorylation. MARCKS phosphorylation and serotonin release from permeabilized platelets have the same time course and were blocked by a peptide (MPSD) with the amino acid sequence corresponding to the phosphorylation site domain of MARCKS. Pleckstrin and myosin light chain phosphorylation was not modified. A peptide (Ala-MPSD) in which the four serine residues of MPSD were substituted by alanines was ineffective. These results provide the first evidence that MARCKS may play a role in platelet secretion. Moreover, pleckstrin phosphorylation has a different time course than that of MARCKS or serotonin release and was not modified when MARCKS phosphorylation and serotonin release were inhibited, suggesting that pleckstrin is either not directly involved in secretion or that it might only be involved upstream in the cascade of events leading to exocytosis.
先前的实验表明，血小板分泌可能需要肌动蛋白在特定部位解聚。佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）刺激血小板可诱导血小板-白细胞C激酶底物（pleckstrin）磷酸化、血小板聚集和分泌。抑制蛋白激酶C（PKC）会伴随着pleckstrin磷酸化和5-羟色胺分泌的抑制。在此，我们证明了富含肉豆蔻酰化丙氨酸的C激酶底物（MARCKS），另一种PKC底物，在血小板中的存在及其在PMA刺激过程中的磷酸化。已知MARCKS可结合肌动蛋白并交联肌动蛋白丝；后者会被PKC诱导的MARCKS磷酸化所抑制。MARCKS磷酸化和通透化血小板中5-羟色胺的释放具有相同的时间进程，并被一种氨基酸序列与MARCKS磷酸化位点结构域相对应的肽（MPSD）所阻断。pleckstrin和肌球蛋白轻链磷酸化未被改变。一种将MPSD的四个丝氨酸残基替换为丙氨酸的肽（Ala-MPSD）无效。这些结果首次证明MARCKS可能在血小板分泌中起作用。此外，pleckstrin磷酸化的时间进程与MARCKS或5-羟色胺释放的时间进程不同，并且当MARCKS磷酸化和5-羟色胺释放被抑制时未被改变，这表明pleckstrin要么不直接参与分泌，要么可能仅在导致胞吐作用的事件级联反应的上游起作用。
