Griffith L C, Schulman H
Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
J Biol Chem. 1988 Jul 5;263(19):9542-9.
Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.
用离子载体A23187、卡巴胆碱或高钾培养基刺激大鼠嗜铬细胞瘤PC12细胞，这些试剂可增加细胞内Ca2+，导致酪氨酸羟化酶磷酸化并激活（诺斯，P.，格里菲思，L.C.，和舒尔曼，H.（1985年）《细胞生物学杂志》101，1182 - 1190）。我们已在PC12细胞中鉴定出三种主要的蛋白激酶，并研究了它们在酪氨酸羟化酶和其他胞质蛋白的Ca2+依赖性磷酸化中的作用。一组PC12蛋白在细胞内Ca2+升高和蛋白激酶C（Ca2+/磷脂依赖性蛋白激酶）激活剂的作用下均发生磷酸化。此外，不同的蛋白组对其中一种或另一种刺激有反应。三种主要的调节激酶，多功能Ca2+/钙调蛋白依赖性蛋白激酶、cAMP依赖性蛋白激酶和蛋白激酶C在体外均能使酪氨酸羟化酶磷酸化。增加Ca2+的试剂或直接激活激酶C的试剂（12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯或1 - 油酰 - 2 - 乙酰甘油）均不会增加cAMP或激活cAMP依赖性蛋白激酶，因此排除了该途径作为这些刺激的介导途径。通过用12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯对PC12细胞进行长期处理来评估蛋白激酶C的作用，该处理会导致其“脱敏”。在以这种方式预处理的细胞中，增加Ca2+内流 的试剂继续最大程度地刺激酪氨酸羟化酶磷酸化，而蛋白激酶C激活剂则完全无效。将三种蛋白激酶在体外使酪氨酸羟化酶磷酸化的胰蛋白酶肽图与体内酪氨酸羟化酶磷酸化产生的磷酸肽图进行比较，结果表明Ca2+/钙调蛋白依赖性激酶的磷酸化最接近体内磷酸化模式。这些结果表明，多功能Ca2+/钙调蛋白依赖性蛋白激酶通过激素和电刺激介导PC12细胞中酪氨酸羟化酶的磷酸化，这些刺激会升高细胞内Ca2+。
