Peters H I, Chang Y W, Traugh J A
Department of Biochemistry, University of California, Riverside 92521, USA.
Eur J Biochem. 1995 Dec 1;234(2):550-6. doi: 10.1111/j.1432-1033.1995.550_b.x.
Phosphorylation of the alpha, beta and delta subunits of elongation factor (EF) 1 by protein kinase C results in stimulation of elongation activity up to threefold both in vivo and in vitro [Venema, R. C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 11,993-11,998, Venema, R. C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 12,574-12,580]. The alpha subunit catalyzes the GTP-dependent binding of amino-acyl-tRNA to the ribosome, while the beta gamma and delta subunits of EF-1 catalyze exchange of the residual GDP on EF-1 alpha for GTP. To determine whether the change in elongation rate following phosphorylation by protein kinase C is due to stimulation of GDP/GTP exchange activity, EF-1 and EF-1.valyl-tRNA-synthetase have been purified from rabbit reticulocytes, phosphorylated in vitro by protein kinase C and the effect of phosphorylation on nucleotide-exchange activity analyzed. The alpha, beta and delta subunits are phosphorylated only on serine, and phosphopeptide maps show distinct phosphopeptides for each subunit. Following quantitative phosphorylation of EF-1 by protein kinase C on the alpha, beta, and delta subunits, a twofold enhancement of the rate of nucleotide exchange over the non-phosphorylated controls is observed with EF-1 and EF-1.valyl-tRNA synthetase. Stimulation of nucleotide exchange results in a two-fold increase in the formation of EF-1 alpha.GTP.Phe-tRNA, leading to an increased rate of binding of Phe-tRNA to ribosomes. The magnitude of stimulation of the exchange rate is similar to that reported previously for the rate of elongation following phosphorylation of EF-1 by protein kinase C. Thus, the enhancement of EF-1 activity in response to 4 beta-phorbol 12-myristate 13-acetate appears to be due to stimulation of the rate of GDP/GTP exchange following phosphorylation of EF-1 by protein kinase C.
蛋白激酶C对延伸因子（EF）1的α、β和δ亚基进行磷酸化，在体内和体外均可使延伸活性增强达三倍之多[维内马，R.C.，彼得斯，H.I.和特劳，J.A.（1991年）《生物化学杂志》266卷，11993 - 11998页，维内马，R.C.，彼得斯，H.I.和特劳，J.A.（1991年）《生物化学杂志》266卷，12574 - 12580页]。α亚基催化氨酰 - tRNA与核糖体的GTP依赖性结合，而EF - 1的βγ和δ亚基催化EF - 1α上残留的GDP与GTP的交换。为了确定蛋白激酶C磷酸化后延伸速率的变化是否归因于GDP / GTP交换活性的刺激，已从兔网织红细胞中纯化出EF - 1和EF - 1·缬氨酰 - tRNA合成酶，在体外由蛋白激酶C进行磷酸化，并分析磷酸化对核苷酸交换活性的影响。α、β和δ亚基仅在丝氨酸上被磷酸化，磷酸肽图谱显示每个亚基有不同的磷酸肽。在蛋白激酶C对EF - 1的α、β和δ亚基进行定量磷酸化后，观察到EF - 1和EF - 1·缬氨酰 - tRNA合成酶的核苷酸交换速率比未磷酸化的对照提高了两倍。核苷酸交换的刺激导致EF - 1α·GTP·苯丙氨酰 - tRNA的形成增加两倍，从而导致苯丙氨酰 - tRNA与核糖体结合速率增加。交换速率的刺激幅度与先前报道的蛋白激酶C对EF - 1磷酸化后延伸速率的幅度相似。因此，响应4β - 佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯时EF - 1活性的增强似乎是由于蛋白激酶C对EF - 1磷酸化后GDP / GTP交换速率的刺激。
