Association of initiation factor eIF-4E in a cap binding protein complex (eIF-4F) is critical for and enhances phosphorylation by protein kinase C.
Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F.
Tuazon
P T
PT
Department of Biochemistry, University of California, Riverside 92521.
Morley
S J
SJ
Dever
T E
TE
Merrick
W C
WC
Rhoads
R E
RE
Traugh
J A
JA
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D007700:Kinetics; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000097:blood; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D010710:Phosphates; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate