Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cδ
Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cδ
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DOI：
10.1074/jbc.m405137200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Miller, KW
中科院分区：
文献类型：
--
作者：
Das, J;Addona, GH;Miller, KW
蛋白激酶C（Protein Kinase C，PKC）是一种重要的信号转导蛋白，其富含半胱氨酸的二酰甘油/佛波酯结合结构域C1是C1 A和C1 B亚结构域的串联重复序列，与全身麻醉药相互作用。为了验证这一假设，我们表达，纯化，并表征了高亲和力的佛波醇结合亚结构域，C1 B，小鼠蛋白激酶Cdelta，并研究了其与全身麻醉醇的相互作用。当荧光佛波醇酯，sapinoxin-D，结合PKC δ C1 B在缓冲液中的摩尔比为1：2，其荧光发射最大值，λ（max），从437位移到425 nm。全身麻醉的醇类，丁醇和辛醇，以浓度依赖性的、可饱和的方式进一步使PKC δ C1 B结合的sapinoxin-D的λ（max）移动到类似于415 nm，表明醇类在离散的变构结合位点相互作用。为了鉴定该位点，PKCdeltaC 1B用三种可光活化的二氮杂环丙烯醇类似物3-azioctanol、7-azioctanol和3-azibutanol进行光标记。质谱分析表明，在PKCdeltaC 1B中的标记馏分中，所有三种醇的化学计量比为1：1。将光标记的PKCdeltaC 1B进行胰蛋白酶消化，通过在线色谱分离片段并通过质谱进行测序。每种偶氮醇在Tyr-236处光掺入。检查PKCdeltaC 1B的已知结构表明，该残基位于佛波酯结合口袋附近，并且在结合佛波酯的大约10埃范围内。本研究结果为蛋白激酶C上的变构麻醉位点提供了直接证据。
Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/ phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1: 2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to similar to415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdeltaC1B was photolabeled with three photoactivable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdeltaC1B at a stoichiometry of 1: 1 in the labeled fraction. The photolabeled PKCdeltaC1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdeltaC1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within similar to10 Angstrom of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.