Identification and cloning of centaurin-alpha - A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain
Identification and cloning of centaurin-alpha - A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain
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DOI：
10.1074/jbc.271.31.18859
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发表时间：
1996-08-02
影响因子：
4.8
通讯作者：
Theibert, AB
中科院分区：
文献类型：
--
作者：
HammondsOdie, LP;Jackson, TR;Theibert, AB
利用基于肌醇1,3,4,5-四聚磷酸磷脂类似物（InsP(4)）的亲和树脂和光亲和标记，我们从大鼠大脑中分离、表征并克隆了一个46 kda的蛋白，我们将其命名为centaurin-alpha。结合特异性通过1-O-[H-3]（3-[4-苯甲酰二氢肉桂基]丙基）- insp(4)光亲和标记确定。Centaurin-alpha对磷脂酰肌醇3,4,5-三磷酸（PtdInsP(3)）的亲和力最高（IC50 = 120 nm），而与5-、12-和>结合的InsP(4)、PtdInsP(2)和InsP(3)的亲和力分别低50倍。利用色氨酸部分氨基酸序列生成的聚合酶链反应产物筛选大鼠脑cDNA文库，获得全长克隆。该2450碱基对cDNA包含一个开放阅读框（ORF），编码419个氨基酸的新蛋白。Northern分析揭示了一个2.5千碱基的转录本在大脑中高度表达。该序列包含一个新的锌指基序，10个锚蛋白样重复序列，并与最近发现的酵母和哺乳动物Arf gtpase激活蛋白具有同源性。考虑到在脑内结合和富集的特异性，centaurin-alpha是一种候选的PtdInsP(3)受体，可能将磷酸肌苷激酶3-激酶的激活与脑内的下游反应联系起来。
Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP(4)), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha. Binding specificity was determined using displacement of 1-O-[H-3](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP(4) photoaffinity labeling. Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) (IC50 = 120 nm), whereas InsP(4), PtdInsP(2), and InsP(3) bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP(3) receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.