Abstract
Valyl-tRNA synthetase from mammalian cells is isolated in a high Mr complex with elongation factor 1 (EF-1). This complex, which represents all of the valyl-tRNA synthetase activity and a significant portion of the EF-1 activity in rabbit reticulocytes, contains five polypeptides identified as valyl-tRNA synthetase and the four subunits of EF-1. In this study, we have examined the potential for regulation of the complex by phosphorylation of these components. The valyl-tRNA synthetase.EF-1 complex has been purified by gel filtration and tRNA-Sepharose chromatography from 32P-labeled rabbit reticulocytes stimulated by phorbol 12-myristate 13-acetate (PMA) and compared to the complex purified from control cells. One- and two-dimensional polyacrylamide gel electrophoresis and autoradiography show that valyl-tRNA synthetase and the alpha, beta and delta subunits of EF-1 are phosphorylated in vivo. Phosphorylation of each of the four proteins is increased 2-4-fold in response to PMA. Phosphorylation of valyl-tRNA synthetase in response to PMA is reproducibly accompanied by a 1.7-fold increase in aminoacylation activity, whereas phosphorylation of EF-1 is associated with a 2.0-2.2-fold stimulation of activity, as measured by poly(U)-directed polyphenylalanine synthesis. These data suggest that stimulation of translational rates in response to PMA is mediated, at least in part, by phosphorylation of valyl-tRNA synthetase and EF-1.
Highlights
Lated in a high M, complex with elongation factor 1 This complex contains three separatecatalytic activities, each (EF-1).This complex, which represeanltlsof thevalyl- of which is potentially rate-limiting in the elongation phase
Phosphorylationof translational components by protein kinase C hasthus been implicated in thecoordinate regulation of protein biosynthesis (16).Phorbol ester treatment habseen shown to increase the rate of translation in intact cells, both at thelevel of gene transcription (17) andby short-term postcomplex containing allof the tRNAsynthetases andpossibly transcriptional mechanisms (18-21), which have been shown other translational components as well
The results presented in this paper provide the first evidence that phorbol esters stimulatephosphorylation of valyltRNA synthetase and EF-1 i n uiuo
Summary
Proteins tentatively identified as the a , @a,nd 6 subunits of EF-1 in the valyl-tRNA synthetase. Most of the Coomassie-stained protein corresponding to tph,ey, and 6 subunits of EF-1 coeluted with the valyl-tRNA synthetase activity. Fractions from the gel filtration column containing valyltRNA synthetase activity were pooled, and the valyl-tRNA synthetase.EF-1 complex was purified further by affinity. As shown by the Coomassie-stained gel (Fig. and PMA-treated samplewsere compared intwo experiments, 3C), the complex was highly purified (>go% pure) and conthe activity of EF-1 associated with valyl-tRNA synthetase tained the five polypeptides identifiedpreviously as valylwas increased by 120% (Fig. 1B) and 96% inresponseto tRNA synthetase and thefour subunits of EF-1 ( 3 ,4). 3 and anti-eIF-4B antibodies (data not shown) Phosphorylation of these two proteins was stimulated by PMA.
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