Modulation of rabbit reticulocyte guanine nucleotide exchange factor activity by casein kinases 1 and 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-kDa subunit of eIF-2B by CK-1 and glycogen synthase kinase 3 (GSK-3) and the effects of this covalent modification on nucleotide exchange. Phosphorylation 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 activity by a factor of 4, and rephosphorylation with CK-1 (0.49 mol of phosphate/mol of eIF-2B) restores its specific activity to that of the phosphorylated protein. GSK-3 phosphorylates the 82-kDa subunit of both isolated and alkaline phosphatase-treated eIF-2B; however, the stoichiometry 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. Phosphorylation 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 protein. 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.
Singh
L P
LP
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216, USA.
Denslow
N D
ND
Wahba
A J
AJ
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D047390:Casein Kinase II; D047388:Casein Kinases; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes
D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3