Regulation of elongation factor-2 by multisite phosphorylation


Correspondence to C. G. Proud, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, England
Fax:+ 44 0272 303497.


We have studied the phosphorylation of protein synthesis elongation factor eEF-2, the effects of phosphorylation on its activity and dephosphorylation of phosphorylated eEF-2 by protein phosphatases-2A and -2C. Extensive analysis of phosphopeptides generated from eEF-2 phosphorylated in vitro, by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates: in this system mono-phosphorylated eEF-2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono-phosphorylated eEF-2 detected are phosphorylated at Thr56.

The eEF-2 kinase could phosphorylate a synthetic peptide based on residues 49–60 of eEF-2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high Km, compared to eEF-2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53.

In the light of the existence of two phosphorylation sites in eEF-2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)-directed synthesis of polyphenylalanine, where both the bis- and mono-phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non-phosphorylated eEF-2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF-2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained.

Protein phosphatases-2A and -2C (PP-2A and PP-2C) can each efficiently dephosphorylate phosphorylated eEF-2. While bis-phosphorylated eEF-2 was a better substrate for PP-2A than mono-phosphorylated factor (phosphorlated at Thr56), the converse was true for PP-2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP-2C by the presence of phosphate on Thr58. Nevertheless, PP-2C exhibited a preference for dephosphorylation of Thr56 in bis-phosphorylated eEF-2, while PP-2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases.