Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F.
Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.
Donaldson
R W
RW
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee.
Hagedorn
C H
CH
Cohen
S
S
D000596:Amino Acids / Q000032:analysis; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D039562:Eukaryotic Initiation Factor-4F; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D019319:Okadaic Acid; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests
D000596:Amino Acids; D004988:Ethers, Cyclic; D039562:Eukaryotic Initiation Factor-4F; D007476:Ionophores; D010448:Peptide Initiation Factors; D019319:Okadaic Acid; D004815:Epidermal Growth Factor