MODULATION OF RABBIT RETICULOCYTE GUANINE-NUCLEOTIDE EXCHANGE FACTOR ACTIVITY BY CASEIN KINASE-1 AND KINASE-2 AND GLYCOGEN-SYNTHASE KINASE-3

The in vitro phosphorylation of the guanine nucleotide exchange factor (eIF-2B) by casein kinase 2 (CK-2) was previously shown to stimulate the binding of GTP to eIF-2B and increase nucleotide exchange [Singh, L. P., Aroor, A, R., & Wahba, A, J, (1994) Biochemistry 33, 9152-9157] , The present study examines the in vitro phosphorylation of the 82-kD a subunit of eIF-2B by CK-1 and glycogen synthase kinase 3 (GSK-3) and the effects of this covalent modification on nucleotide exchange, Pho sphorylation with CK-1 adds approximately 0.27 mol of phosphate/mol of eIF-2B and doubles guanine nucleotide exchange activity. Treatment of the phosphorylated eIF-2B with alkaline phosphatase reduces its activ ity by a factor of 4, and rephosphorylation with CK-1 (0.49 mol of pho sphate/mol of eIF-2B) restores its specific activity to that of the ph osphorylated protein. GSK-3 phosphorylates the: 82-kDa subunit of both isolated and alkaline phosphatase-treated eIF-2B; however, the stoich iometry of phosphorylation is much less (approximately 0.12 mol/mol of eIF-2B in both preparations) than that obtained with CK-1 or CK-2. Ph osphorylation of eIF-2B with GSK-3 neither stimulates nor inhibits GDP /GTP exchange. The results of this study indicate that phosphorylation of eIF-2B with CK-1 and/or CK-2 is required for GTP binding to the pr otein. Evidence is also presented for a mechanism of regulation of eIF -2B activity whereby phosphorylation by GSK-3 influences the activity of the protein and partially suppresses phosphorylation by CK-1 or CK- 2.