Kazanietz M G, Areces L B, Bahador A, Mischak H, Goodnight J, Mushinski J F, Blumberg P M
Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, Bethesda, Maryland 20892.
Mol Pharmacol. 1993 Aug;44(2):298-307.
Analysis of [3H]phorbol-12,13-dibutyrate (PDBu) binding was performed with protein kinase C (PKC)-alpha, -beta 1, -gamma, -delta, -epsilon, -eta, and -zeta produced in Sf9 insect cells using the baculovirus expression system. With the exception of PKC-zeta, all of the PKC isozymes bound [3H]PDBu with high affinity (Kd < 1 nM), either in the presence or in the absence of calcium. Scatchard analysis using 100% phosphatidylserine vesicles revealed slightly lower affinity for the calcium-independent isozymes (PKC-delta, -epsilon, and -eta) than for the calcium-dependent isozymes (PKC-alpha, -beta, and -gamma). Competition for [3H]PDBu binding by different classes of PKC activators showed that 12-deoxyphorbol esters, mezerein, and octahydromezerein likewise possessed lower affinity for the calcium-independent isozymes. The mezerein analog thymeleatoxin was the most marked example, being almost 20-fold less potent for binding to PKC-epsilon and -eta than to PKC-beta 1. In contrast, the indole alkaloids (-)-indolactam V and (-)-octylindolactam V and the postulated endogenous activator 1,2-diacylglycerol bound with similar affinities to all of the PKC isoforms, suggesting that different residues/configurations in the binding sites of the different PKC isozymes might be involved in interaction with the pharmacophore of the activators. The seven PKC isozymes also showed clearly different substrate specificities with exogenous peptide and protein substrates. The heterogeneous behavior of the different members of the PKC family with ligands and substrates may contribute to the heterogeneity of PKC-mediated pathways at the cellular level.
使用杆状病毒表达系统，对在Sf9昆虫细胞中产生的蛋白激酶C（PKC）-α、-β1、-γ、-δ、-ε、-η和-ζ进行了[3H]佛波醇-12,13-二丁酸酯（PDBu）结合分析。除PKC-ζ外，所有PKC同工酶在有或无钙的情况下均以高亲和力（Kd < 1 nM）结合[3H]PDBu。使用100%磷脂酰丝氨酸囊泡进行的Scatchard分析表明，与钙依赖性同工酶（PKC-α、-β和-γ）相比，钙非依赖性同工酶（PKC-δ、-ε和-η）的亲和力略低。不同类别的PKC激活剂对[3H]PDBu结合的竞争表明，12-脱氧佛波醇酯、芫花酯素和八氢芫花酯素同样对钙非依赖性同工酶具有较低的亲和力。芫花酯素类似物百里香毒素是最明显的例子，其与PKC-ε和-η结合的效力比对PKC-β1低近20倍。相比之下，吲哚生物碱（-）-吲哚内酰胺V和（-）-八氢吲哚内酰胺V以及假定的内源性激活剂1,2-二酰基甘油与所有PKC同工型的结合亲和力相似，这表明不同PKC同工酶结合位点中的不同残基/构型可能参与了与激活剂药效基团的相互作用。这七种PKC同工酶对外源肽和蛋白质底物也表现出明显不同的底物特异性。PKC家族不同成员与配体和底物的异质性行为可能导致细胞水平上PKC介导途径的异质性。
