Phorbol esters stimulate phosphorylation of eukaryotic initiation factors 3, 4B, and 4F.
Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, was isolated by m7 GTP-Sepharose affinity chromatography from rabbit reticulocytes incubated with [32P]orthophosphate. Following treatment of reticulocytes with phorbol 12-myristate 13-acetate (PMA) for 30 min, stimulation of phosphorylation of both the p25 and p220 subunits was observed (2.5-5-fold). Two variants were observed for p25 in the absence and presence of PMA when analyzed by two-dimensional gel electrophoresis. Only the more acidic of these was phosphorylated, with the level of phosphorylation increased upon PMA treatment. One main variant was observed for p220; following PMA stimulation, in addition to increased labeling of this variant, two more acidic phosphorylated variants were observed. Low levels of eIF-3 and -4B were associated with purified eIF-4F, and PMA treatment stimulated phosphorylation of eIF-3 (p170) by 2-4-fold and eIF-4B by 1.5-2.5 fold. Two-dimensional phosphopeptide mapping of p25 phosphorylated in the absence or presence of PMA generated a single tryptic phosphopeptide, suggesting a single phosphorylation site. A more complex phosphopeptide map was observed with p220 subunit. The maps for both subunits contained the same phosphopeptides as those obtained when eIF-4F was phosphorylated in vitro by the Ca2+/phospholipid-dependent protein kinase, indicating this protein kinase directly modulated eIF-4F in response to PMA.
Morley
S J
SJ
Department of Biochemistry, University of California, Riverside 92521.
Traugh
J A
JA
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D039621:Eukaryotic Initiation Factor-3; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D001426:Bacterial Proteins; D039621:Eukaryotic Initiation Factor-3; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D010710:Phosphates; D010748:Phosphopeptides; C031799:eIF-4B; D013755:Tetradecanoylphorbol Acetate