Chang Y W, Traugh J A
Department of Biochemistry, University of California, Riverside 92521, USA.
Eur J Biochem. 1998 Jan 15;251(1-2):201-7. doi: 10.1046/j.1432-1327.1998.2510201.x.
To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-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-1alpha 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 alpha 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 in vitro; the remainder were unique. To examine whether protein kinase C had 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 4beta-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].
为研究延伸因子eEF-1的磷酸化在翻译调控中的作用，用³²P标记3T3-L1细胞，使其血清饥饿，然后在有或无10 nM胰岛素的情况下孵育15分钟。通过tRNA-琼脂糖亲和层析纯化eEF-1，结果显示其α、β和δ亚基均发生了磷酸化。胰岛素刺激后，eEF-1α的磷酸化增加了6倍，β增加了4倍，δ增加了3倍。用来自胰岛素刺激细胞的eEF-1测定的延伸速率比血清饥饿细胞的eEF-1高出两倍多。对胰岛素处理细胞的eEF-1进行二维胰蛋白酶磷酸肽图谱分析，α亚基得到9个磷酸肽，β亚基得到1个，δ亚基得到3个。与胰岛素刺激的多潜能蛋白激酶体外磷酸化的eEF-1的α、β和δ亚基的磷酸肽图谱相比，β和δ亚基的图谱相同。体内α亚基获得的5个磷酸肽与体外S6激酶获得的相同；其余的是独特的。为研究蛋白激酶C是否在胰岛素刺激的eEF-1磷酸化中起作用，通过将3T3-L1细胞长期暴露于4β-佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）来下调蛋白激酶C。胰岛素刺激后，α、β和δ亚基的磷酸化增加了2.5倍，延伸活性也受到类似程度的刺激，这表明蛋白激酶C对胰岛素刺激的延伸没有影响。因此，胰岛素刺激的eEF-1活性似乎主要由多潜能S6激酶介导。该数据与先前关于多潜能S6激酶通过起始因子磷酸化刺激起始的研究一致[Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989]。
