The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes
The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes
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DOI：
10.1074/jbc.m204681200
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发表时间：
2002-09-13
影响因子：
4.8
通讯作者：
Tavaré, JM
中科院分区：
文献类型：
--
作者：
Berwick, DC;Hers, I;Tavaré, JM
蛋白激酶B（Akt）在细胞调控中起着核心作用，尽管该激酶的许多生理学相关底物仍有待鉴定。在这项研究中，我们从原代附睾脂肪细胞中分离出一种表观分子量为125，000的蛋白质。该蛋白以胰岛素依赖性方式与针对蛋白激酶B底物共有序列RXRXX（pS/pT）的磷酸化特异性抗体以及识别GSK-3 α/β的丝氨酸21/9的磷酸化特异性抗体表现出免疫反应性。MALDI-TOF质谱显示的蛋白质是ATP-柠檬酸裂解酶，这表明两个磷酸特异性抗体识别磷酸丝氨酸454，以前报道的胰岛素和异丙肾上腺素刺激的ATP-柠檬酸裂解酶磷酸化位点。事实上，胰岛素和异丙肾上腺素刺激磷酸化的磷酸化特异性抗体识别的网站上的这种蛋白质在渥曼青霉素敏感和不敏感的方式，分别。此外，在原代脂肪细胞中组成型活性蛋白激酶B的瞬时表达模拟了胰岛素对ATP-柠檬酸裂解酶磷酸化的影响。此外，ATP-柠檬酸裂合酶在体外被重组蛋白激酶B在同一位点磷酸化。综上所述，这些结果表明ATP-柠檬酸裂解酶的丝氨酸454是蛋白激酶B的一种新的和主要的体内底物。
Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3alpha/beta. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and major in vivo substrate for protein kinase B.