Sarafian T, Pradel L A, Henry J P, Aunis D, Bader M F
Institut National de la Santé et de la Recherche Medicale Unité-338 de Biologie de la Communication Cellulaire, Strasbourg, France.
J Cell Biol. 1991 Sep;114(6):1135-47. doi: 10.1083/jcb.114.6.1135.
Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.
经通透处理的肾上腺嗜铬细胞在微摩尔钙浓度刺激下通过胞吐作用分泌儿茶酚胺。最近，我们已经证明，用洋地黄皂苷通透处理的嗜铬细胞由于释放了胞吐作用所必需的某些胞质蛋白而逐渐丧失其分泌能力（萨拉菲安T.、D. 奥尼斯和M. F. 巴德。1987年。《生物化学杂志》34:16671 - 16676）。在此我们表明，释放的蛋白之一是钙结合蛋白I，一种钙依赖性磷脂结合蛋白，已知在生理微摩尔钙水平促进嗜铬颗粒的体外聚集。将钙结合蛋白I添加到因胞质蛋白泄漏而分泌能力降低的经洋地黄皂苷或链球菌溶血素O通透处理的嗜铬细胞中，可部分恢复钙依赖性分泌活性。这种效应是钙结合蛋白I特有的，因为其他膜联蛋白（p32、p37、p67）在相似或更高浓度下不会刺激分泌。钙结合蛋白I需要Mg - ATP的存在，这表明磷酸化步骤可能调节钙结合蛋白的活性。钙结合蛋白无法恢复完全丧失胞质蛋白激酶C的细胞或蛋白激酶C被鞘氨醇抑制或通过与佛波酯长期孵育而下调的细胞中的分泌活性。相反，经纯化的蛋白激酶C体外预磷酸化的钙结合蛋白I能够在缺乏蛋白激酶C活性的细胞中重建分泌。用对细胞磷酸酶有抗性的硫代磷酸化钙结合蛋白I或在磷酸酶抑制剂微囊藻毒素存在下引入细胞的磷酸化钙结合蛋白I也观察到这种刺激效应。这些结果表明，钙结合蛋白I通过一种需要蛋白激酶C磷酸化的机制参与胞吐机制。
