Terbush D R, Holz R W
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0626.
J Biol Chem. 1990 Dec 5;265(34):21179-84.
We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.
我们通过使用PKC(19 - 31)（一种蛋白激酶C假底物抑制肽）、Ca/CaM激酶II(291 - 317)（一种钙调蛋白结合肽）和星形孢菌素，研究了蛋白激酶C激活在嗜铬细胞儿茶酚胺胞吐作用中是起调节作用还是必不可少的作用。在透化细胞中，PKC(19 - 31)可将佛波酯介导的Ca2(+)依赖性分泌增强抑制多达90%，但在无佛波酯时对Ca2(+)依赖性分泌无影响。PKC(19 - 31)对佛波酯诱导的分泌增强的抑制与该肽抑制原位佛波酯刺激的蛋白激酶C活性的能力密切相关。PKC(19 - 31)还可阻断12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯(TPA)诱导的透化细胞中多种内源性蛋白的磷酸化，但对Ca2(+)刺激的酪氨酸羟化酶磷酸化无影响。源自Ca/钙调蛋白激酶II钙调蛋白结合区域的Ca/CaM激酶II(291 - 317)，在有或无佛波酯存在时对Ca2(+)依赖性分泌均无影响。该肽完全阻断了Ca2(+)依赖性的酪氨酸羟化酶磷酸化增加，但对透化细胞中TPA诱导的内源性蛋白磷酸化无影响。为了确定分泌是否可能需要一种长寿命的蛋白激酶C底物，在对完整细胞进行透化和测量分泌之前，将亲脂性蛋白激酶抑制剂星形孢菌素加入完整细胞中30分钟。星形孢菌素强烈抑制佛波酯介导的Ca2(+)依赖性分泌增强。在无佛波酯时，它对Ca2(+)依赖性分泌有轻微抑制作用，这一抑制作用不易归因于对蛋白激酶C的抑制。星形孢菌素还抑制佛波酯介导的完整细胞中高钾诱导分泌的增强，同时增强45Ca2+摄取。在无TPA时，星形孢菌素对高钾刺激的分泌有轻微抑制作用。数据表明，蛋白激酶C的激活在胞吐毒性途径中起调节作用而非必不可少的作用。
