Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in polypeptide chain initiation.
We have demonstrated that the purified guanine nine nucleotide exchange factor (GEF) may be isolated as a complex with NADPH. Complete inhibition of the GEF-catalyzed exchange of eukaryotic initiation factor 2-bound GDP for GTP was observed in the presence of either 0.5-0.75 mM NAD+ or NADP+. Incubation of GEF with ATP results in the phosphorylation of its Mr 82,000 polypeptide. This phosphorylation is strongly inhibited by heparin but is not affected by heme or H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP- and cGMP-dependent protein kinases and protein kinase C. The purification of GEF was modified to eliminate any contaminating kinase activity and the isolated protein appears to be homogeneous as judged by NaDodSO4/polyacrylamide gel electrophoresis and silver staining. The Mr 82,000 subunit of GEF is phosphorylated only upon addition of ATP and casein kinase II. The extent of phosphorylation is approximately equal to 0.55 mol of phosphate per mol of GEF, and this results in a 2.3-fold increase in the guanine nucleotide exchange activity. Following treatment of the phosphorylated GEF with alkaline phosphatase, the activity of the protein is reduced by a factor of 5. Rephosphorylation of GEF increases its specific activity to that of the phosphorylated protein. The results of this study suggest that phosphorylation/dephosphorylation of GEF plays a role in regulating polypeptide chain initiation.
Dholakia
J N
JN
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Wahba
A J
AJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000469:Alkaline Phosphatase