Liu J P, Engler D, Funder J W, Robinson P J
Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia.
Mol Cell Endocrinol. 1994 Nov;105(2):217-26. doi: 10.1016/0303-7207(94)90173-2.
We have recently shown that AVP causes a protein kinase C (PKC)-dependent increase in ACTH release and biosynthesis in ovine anterior pituitary cells. In these cells, AVP also causes the translocation of PKC from the cytosol to the cell membrane which is maximal at 5 min, but the intracellular events distal to protein kinase C activation that underlie ACTH secretion have not been well characterized to date. Since the MARCKS protein has been implicated in neurosecretion and is phosphorylated by PKC in synaptosomes, studies were carried out to determine whether AVP might cause MARCKS phosphorylation in the ovine anterior pituitary, and to determine whether this phenomenon might be temporally correlated with PKC translocation and the release of ACTH. When cytosolic fractions of rat brain, ovine anterior pituitary, and cultured ovine anterior pituitary cells were incubated with purified PKC, several proteins were phosphorylated including those in the region of 83-85 kDa. After precipitation of the proteins with 40% acetic acid, the 83-85 kDa phosphoproteins were selectively recovered in the acid soluble phase. Phosphopeptide maps of either the 83 or 85 kDa proteins were generated with Staphylococcus aureus V8 protease and revealed 13 and 9 kDa phosphopeptides, which are characteristic of the authentic MARCKS protein. An identical phosphopeptide map was also obtained when the MARCKS protein was selectively extracted from intact 32P-labeled anterior pituitary cells. MARCKS phosphorylation was markedly increased when ovine anterior pituitary cells were exposed to 1 microM phorbol 12-myristate 13-acetate (PMA). When the cells were exposed to 1 microM AVP, MARCKS phosphorylation increased at 15 s and reached the maximal plateau value at 30 s. MARCKS phosphorylation then started to diminish at 2 min, and baseline levels were attained by 10 min. In the same cells, AVP stimulated ACTH release in a biphasic manner-during the first 30 s, there resulted a rapid burst of ACTH secretion that was followed by a slower, but sustained rate of secretion. We conclude that: (1) AVP causes a rapid, and reversible, phosphorylation of the MARCKS protein in the ovine anterior pituitary; (2) since the AVP-induced increase in MARCKS phosphorylation occurs much earlier in these cells than does PKC trans-location, MARCKS phosphorylation may provide a more sensitive index of the onset of PKC activation than the translocation assay; (3) the close temporal association between MARCKS phosphorylation and the rapid early release of ACTH suggests that MARCKS phosphorylation may be involved in the initial intracellular events that underly exocytosis of the hormone.
我们最近发现，血管加压素（AVP）可使绵羊垂体前叶细胞中促肾上腺皮质激素（ACTH）的释放及生物合成增加，且该作用依赖蛋白激酶C（PKC）。在这些细胞中，AVP还可使PKC从胞质溶胶转位至细胞膜，此转位在5分钟时达到最大值，但迄今为止，PKC激活后导致ACTH分泌的细胞内远端事件尚未得到充分表征。由于富含丙氨酸的豆蔻酰化蛋白激酶C底物（MARCKS）蛋白与神经分泌有关，且在突触体中可被PKC磷酸化，因此我们开展了研究，以确定AVP是否会导致绵羊垂体前叶中MARCKS磷酸化，并确定这种现象是否可能与PKC转位及ACTH释放存在时间上的相关性。当将大鼠脑、绵羊垂体前叶及培养的绵羊垂体前叶细胞的胞质部分与纯化的PKC一起孵育时，几种蛋白质发生了磷酸化，包括分子量在83 - 85 kDa区域的蛋白质。用40%乙酸沉淀蛋白质后，83 - 85 kDa的磷蛋白选择性地存在于酸溶性相中。用金黄色葡萄球菌V8蛋白酶生成了83 kDa或85 kDa蛋白质的磷酸肽图谱，显示出13 kDa和9 kDa的磷酸肽，这是真实MARCKS蛋白的特征。当从完整的32P标记的垂体前叶细胞中选择性提取MARCKS蛋白时，也获得了相同的磷酸肽图谱。当绵羊垂体前叶细胞暴露于1 μM佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）时，MARCKS磷酸化显著增加。当细胞暴露于1 μM AVP时，MARCKS磷酸化在15秒时增加，并在30秒时达到最大平台值。然后，MARCKS磷酸化在2分钟时开始下降，并在10分钟时恢复到基线水平。在同一细胞中，AVP以双相方式刺激ACTH释放——在前30秒内，ACTH分泌迅速爆发，随后是较慢但持续的分泌速率。我们得出以下结论：（1）AVP可使绵羊垂体前叶中的MARCKS蛋白快速且可逆地磷酸化；（2）由于在这些细胞中，AVP诱导的MARCKS磷酸化比PKC转位发生得更早，因此与转位测定相比，MARCKS磷酸化可能是PKC激活起始的更敏感指标；（3）MARCKS磷酸化与ACTH的快速早期释放之间密切的时间关联表明，MARCKS磷酸化可能参与了该激素胞吐作用的初始细胞内事件。
