Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity
Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity
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DOI：
10.1046/j.1432-1327.1998.2510201.x
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发表时间：
1998-01-15
期刊：
影响因子：
--
通讯作者：
Traugh, JA
中科院分区：
文献类型：
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作者：
Chang, YWE;Traugh, JA
为了研究延伸因子eEF-1的磷酸化在翻译调节中的作用，将P-32标记的3 T3-L1细胞去除血清，然后在存在或不存在10 nM胰岛素的情况下孵育15 min。通过在tRNA-Sepharose上进行亲和色谱纯化eEF-1，并显示其在α、β和δ亚基上磷酸化。eEF-1 α的磷酸化被胰岛素刺激6倍，β被刺激4倍，δ被刺激3倍。用eEF-1从胰岛素刺激的细胞测定的延伸率是用eEF-1从血清剥夺的细胞测定的延伸率的两倍以上。当eEF-1从胰岛素处理的细胞进行二维胰蛋白酶磷酸肽映射，获得9个磷酸肽与a亚基，一个与β亚基和三个与δ亚基。当与eEF-1的α，β和δ亚基在体外由胰岛素刺激的多能蛋白激酶磷酸化的磷酸肽图谱相比时，β和δ亚基的图谱是相同的。五个磷酸肽与α亚基在体内获得的S6激酶III体外相同，其余的是独特的。为了检测蛋白激酶C是否在响应胰岛素的eEF-1磷酸化中起作用，通过将3 T3-L1细胞长期暴露于4 β-佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）来下调蛋白激酶C。磷酸化的α，β和δ亚基刺激2.5倍，响应于胰岛素，延长活动刺激到类似的程度：表明蛋白激酶C没有影响刺激响应于胰岛素的延长。因此，胰岛素刺激eEF-1活性似乎主要由多能S6激酶介导。该数据与先前关于通过多能S6激酶磷酸化起始因子来刺激起始的研究一致[莫利，S. J. & Traugh，J. A.（1993）Biochemie（巴黎）95，985-989]。
To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, P-32-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1 alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the a subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase iii vitro; the remainder were unique. To examine whether protein kinase C bad a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4 beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent: suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].