PKCε has an alcohol-binding site in its second cysteine-rich regulatory domain
PKCε has an alcohol-binding site in its second cysteine-rich regulatory domain
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DOI：
10.1042/bj20082271
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发表时间：
2009-08-01
影响因子：
4.1
通讯作者：
Slater, Simon J.
中科院分区：
文献类型：
--
作者：
Das, Joydip;Pany, Satyabrata;Slater, Simon J.
醇类调节蛋白激酶C（PKC）的表达和功能，研究表明醇类结合位点位于PKC α的C1结构域，该结构域由两个富含半胱氨酸的亚结构域C1 A和C1 B组成。与野生型相比，PKC ε基因敲除小鼠的饮酒量显着减少。本研究的目的是探讨是否所有的酒精结合位点都可能存在于PKC β中。我们发现乙醇以浓度依赖性方式抑制PKC β 1活性，EC（50）乙醇、丁醇和辛醇增加了荧光佛波醇酯SAPD的结合亲和力（sapinoxin-D）以浓度依赖性方式对PKC β C1 B具有抑制作用，EC（50）值分别为78 mM、8 mM和340 μ M，提示在该亚结构域中存在所有变构的醇结合位点。为了鉴定该位点，用3-azibutanol和3-azibutanol对PKC β C1 B进行光标记，并通过MS进行分析。而azibutanol优先标记His（236），Tyr（238）是azibutanol的优选位点。对PKC β C1 B的模型结构的检查显示，这些残基彼此相距3.46埃（1埃= 0.1 nm），并形成一个沟槽，其中His（236）暴露在表面，Tyr（238）埋在内部。当这些残基被丙氨酸取代时，它显着降低了突变体H236 A/Y238 A中的光标记和酒精诱导的SAPID结合的酒精结合。而Tyr（228）在突变体H236 A中被标记，His（236）在突变体Y238 A中被标记。本研究结果为油蛋白激酶C β存在非变构的醇结合位点提供了直接证据，并强调了His（236）和Tyr（238）残基在醇结合中的作用。
Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that ail alcohol-binding site is present in PKC alpha in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKC epsilon-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present Study was to investigate whether ail alcohol-binding site could be present in PKC epsilon. Here we show that ethanol inhibited PKC epsilon activity in a concentration-dependent manner with an EC(50) (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKC epsilon C1B in a concentration-dependent manner with EC(50) values of 78 mM, 8 mM and 340 mu M respectively, suggesting the presence of ail allosteric alcohol-binding site ill this subdomain. To identify this site, PKC epsilon C1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His(236), Tyr(238) was the preferred site for azioctanol. Inspection of the model structure of PKC epsilon C1B reveals that these residues are 3.46 angstrom (1 angstrom = 0.1 nm) apart from each other and form a groove where His(236) is surface-exposed and Tyr(238) is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding ill terms of both photolabelling and alcohol-induced SAPID binding in the mutant H236A/Y238A. Whereas Tyr(228) was labelled in mutant H236A, His(236) was labelled in mutant Y238A. The present results provide direct evidence for the presence of,ill allosteric alcohol-binding site oil protein kinase C epsilon and underscore the role of His(236) and Tyr(238) residues in alcohol binding.