Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.
Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.
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人蛋白激酶 C α 和 zeta 同工酶的激活和底物特异性。
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发表时间：
1993
期刊：
影响因子：
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通讯作者：
T. Sarre
中科院分区：
文献类型：
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作者：
G. Kochs;R. Hummel;D. Meyer;H. Hug;D. Marmé;T. Sarre
蛋白激酶C（PKC）是一类由Ca ~（2+）和/或磷脂激活的丝氨酸/苏氨酸激酶，参与细胞增殖、分化和分泌等多种细胞过程。PKC基因家族的9个成员是已知的;它们在真核细胞中差异表达，并且可以分为两个亚组：Ca（2+）依赖性（经典）PKC同工酶α、β I、β II和γ，以及Ca（2+）非依赖性新PKC同工酶δ、β I、ζ、η和θ。这些PKC同工酶的详细生化特性是阐明其在细胞信号转导中的独特作用的先决条件。在这项研究中，我们报告的人PKC-zeta cDNA的克隆，其在重组杆状病毒感染的昆虫细胞中的表达和PKC-zeta同工酶的部分纯化。与高度纯化的人PKC α（经典PKC亚组的代表）相比，纯化的PKC ζ的特征在于激活剂要求、底物特异性、蛋白水解激活和对PKC抑制剂的敏感性。与PKC α相反，PKC zeta表现出独立于Ca 2+、磷脂酰丝氨酸和二酰基甘油的组成性激酶活性。单独的花生四烯酸或γ-亚麻酸和磷脂酰丝氨酸的组合略微增强PKC ζ活性。在经典的PKC激活剂磷脂酰丝氨酸/二酰基甘油的存在下，PKC α磷酸化PKC-α假底物衍生肽、表皮生长因子受体衍生肽、组蛋白III-S和髓鞘碱性蛋白至相同程度，而PKC ζ仅磷酸化PKC-α衍生肽。然而，花生四烯酸大大降低了PKC-α对表皮生长因子受体衍生肽、组蛋白III-S和髓鞘碱性蛋白的活性，但增强了PKC-ζ对PKC-α衍生肽的活性。这些结果表明，这两种PKC同工酶的底物特异性的可能调制（的结合）不同的激活剂（他们的监管结构域）。就PKC zeta而言，这一发现得到了以下事实的加强：表皮生长因子受体衍生肽（不是全酶的底物）被有限蛋白水解产生的蛋白片段显着磷酸化，并且仅包含激酶结构域。此外，与PKC α相反，PKC ζ对已知干扰调节或催化结构域的PKC抑制剂不敏感，并且不能被过表达相应PKC同工酶的NIH 3 T3细胞或昆虫细胞的佛波醇酯处理激活。这些发现的潜在影响的机制（S）的激活和底物特异性的PKC ζ进行了讨论。
Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca(2+)-dependent (classical) PKC isoenzymes alpha, beta I, beta II and gamma, and the Ca(2+)-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.