Regulation of the activity of eukaryotic peptide elongation factor 1 by autocatalytic phosphorylation

Abstract

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [ Y -~~P I G T P and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF-lcr subunit of the factor ( M , 53000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [ Y -~~P I G T P with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32P04, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21 -0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome-independent, acid-labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl-tRNA to 80s ribosomes in the presence of poly(U). This is accompanied by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparently activates its binding site for aminoacyl-tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor-catalyzed binding of aminoacyl-tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF-1 . GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl-tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP-dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis.Several cellular functions in mammalian tissues are modulated by reversible protein phosphorylation, which is considered to be a major general mechanism of metabolic regulation [ 11. Many enzymes and also protein-synthesis factors [2 -41 are phosphorylated by different protein kinases, which bind phosphate groups by ester linkages to serine, threonine and tyrosine residues in the protein, and the resulting bond is acid-stable (see [5] for a review).Autophosphorylation occurring in the absence of kinases represents another mechanism of protein phosphorylation. It results in an acid-labile binding of phosphate to histidine or lysine residues by phosphoamide linkage [6, 71. Such a phosphorylating activity, utilizing phosphate liberated from GTP, has been found to be associated with eIF-2 [8].The prokaryotic EF-Tu, a functional analogue of eukaryotic eEF-1, reveals a significant GTPase activity, which is involved in the el...

The role of cholesteryl 14‐methylhexadecanoate in the function of eukaryotic peptide elongation factor 1

“…Earlier results from o u r laboratory have suggested that there is a close relation between the autocatalytic phosphorylation of eEF-1 and the binding of aminoacyl-tRNA by the factor [7]. Because of the apparent involvement of CMH in the factor phosphorylation, the role of CMH in the binding of aminoacyl-tRNA by eEF-1 indicated in our previous papers [2, 31 was studied from this point of view in our next experiments.…”

“…Earlier experiments have demonstrated that eEF-1 exhibits a ribosome-independent GTPase activity [7, 131 and the released phosphate is utilized for the autophosphorylation of eEF-1 protein [7]. Addition of CMH did only slightly stimulate the GTPase activity of native eEF-1.…”

“…Since the autophosphorylation of eEF-1 is coupled with the GTPase activity of the factor [7] it was not surprising to find that this reaction was again only slightly stimulated by the addition of CMH if native factor was used. However, the time course of phosphorylation in the presence of the ester indicated that the reaction proceeds significantly more rapidly than in mixtures without CMH (Fig.…”

“…This preparation was homogeneous as revealed by polyacrylamide gel electrophoresis under denaturating conditions. Individual subunits of the factor were separated by chromatofocusing in 5 M urea as described by TuhaEkovi et al [7].…”

“…The GTP-dependent autophosphorylation of peptide-initiation [6] and peptide-elongation factors [7] has been demonstrated as an important mechanism regulating the activity of protein-synthesis factors.…”