Chakravarthy B R, Isaacs R J, Morley P, Durkin J P, Whitfield J F
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
J Biol Chem. 1995 Jan 20;270(3):1362-8. doi: 10.1074/jbc.270.3.1362.
Raising the external Ca2+ concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca(2+)- or TPA-stimulated keratinocytes. Raising the external Ca2+ concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca2+ concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. Lopez-Barahona, M., and Aaronson, S. A. (1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca2+ did not. Furthermore, Ca(2+)-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca2+.CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca2+.Ca2+.calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca2+ to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca2+.calmodulin complexes, formed by the internal Ca2+ surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.
将细胞外钙离子浓度从0.05 mM提高到1.8 mM，对BALB/MK小鼠角质形成细胞中膜相关蛋白激酶C（PKC）活性的刺激作用与特异性PKC激活剂12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）一样强烈。这通过从经钙离子或TPA刺激的角质形成细胞中分离出的膜对PKC选择性肽底物Ac - FKKSFKL - NH₂磷酸化增加得以表明。将细胞外钙离子浓度提高到1.8 mM还引发细胞内游离钙离子浓度升高4倍。如其他地方所报道的（莫斯卡，J. 弗莱明，T. P.，莫洛伊，C. J. 洛佩斯 - 巴拉奥纳，M.，以及阿伦森，S. A.（1989年）《生物化学杂志》264卷，第11228 - 11235页），TPA刺激完整角质形成细胞中PKC底物85 kDa肉豆蔻酰化富含丙氨酸的蛋白激酶C底物（MARCKS）蛋白的磷酸化，但钙离子无此作用。此外，钙离子预处理降低了完整细胞中TPA诱导的85 kDa蛋白的磷酸化。在用1.8 mM钙离子刺激之前，用钙离子/钙调蛋白依赖性磷酸酶抑制剂环孢菌素A处理角质形成细胞，MARCKS磷酸化没有显著增加。在存在诸如氟化物、焦磷酸盐和钒酸盐等磷酸酶抑制剂的情况下，钙离子/钙调蛋白在体外抑制分离膜对85 kDa MARCKS全蛋白的磷酸化，并且这种抑制作用可被钙调蛋白拮抗剂钙调蛋白结合结构域肽克服。因此，1.8 mM钙离子在角质形成细胞中强烈刺激膜PKC活性而不刺激MARCKS蛋白磷酸化的能力与由细胞内钙离子激增形成的钙离子/钙调蛋白复合物结合并阻断这种PKC蛋白底物磷酸化的可能性是一致的。
