PHOSPHORYLATION OF RABBIT RETICULOCYTE GUANINE-NUCLEOTIDE EXCHANGE FACTOR IN-VIVO - IDENTIFICATION OF PUTATIVE CASEIN KINASE-II PHOSPHORYLATION SITES

The guanine nucleotide exchange factor (GEF) is a multi-subunit protei n which catalyzes the exchange of GDP for GTP in eukaryotic chain init iation factor 2. Phosphorylation of the 82-kDa subunit of GEF in vitro by casein kinase II (CK-II) is associated with a 5-fold increase in n ucleotide exchange activity. However, phosphorylation of GEF in vivo h as not been studied, and the kinase(s) that phosphorylate GEF have not been identified. The 82-kDa subunit of GEF was partially sequenced, a nd a synthetic peptide was used to generate polyclonal anti-peptide an tibodies that react specifically with this subunit. To examine the pho sphorylation of GEF in intact cells, the protein was isolated and puri fied extensively from metabolically P-32-labeled rabbit reticulocytes. Only the 82-kDa subunit was found to be phosphorylated, and on Wester n blots the anti-peptide antisera reacted specifically with the labele d subunit. Phosphoamino acid analysis indicated that phosphorylation o ccurred exclusively on Ser residues. Digestion with cyanogen bromide o f in vivo labeled protein and GEF phosphorylated in vitro by CK-II pro duced comparable phosphopeptide maps. However, additional phosphopepti de bands were also observed with GEF derived from intact cells. Sequen ce analysis obtained by Edman degradation of the phosphopeptides was c ompared with the deduced amino acid sequence of a cloned 82-kDa subuni t of GEF [Bushman, J. L., Asuru, A. I., Matts, R. L., and Hinnenbusch, A. G. (1993) Mol. Cell, Biol. 13, 1920-1932]. Putative sites of phosp horylation were identified at Ser 703 and/or 704, which contain the se quence S(P)XXD, a CK-II consensus recognition motif. In addition, the Ser residue at position 174 could also be phosphorylated by CK-II in a hierarchical manner. These results suggest that the 82-kDa subunit of GEF is phosphorylated in vivo by CK-II or a similar enzyme, and this covalent posttranslational modification is a potential mechanism for t he regulation of GEF in eukaryotic cells.