Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F.
Initiation factor eIF-4F, a multiprotein cap binding protein complex, was purified from HeLa cells by m7G affinity chromatography and independently by phosphocellulose column chromatography. The m7G affinity-purified sample contains three major proteins, p220, eIF-4A, and p28 (also known as CBP-I or eIF-4E). The abundancies of these proteins are roughly 2, 10, and 0.8 X 10(6) molecules/cell, respectively. Two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eIF-4F samples shows that p28 comprises two isoelectric variants, one of which labels with phosphate and disappears when samples are treated with alkaline phosphatase. The 45,000-dalton protein in eIF-4F appears to be identical to eIF-4A. The p220 subunit rarely produces discrete spots on two-dimensional gel electrophoresis; in purified samples it usually forms 3 closely spaced streaks. eIF-4F fractionated by phosphocellulose chromatography separates into forms containing either phosphorylated or unphosphorylated p28. However, both fractions possess similar specific activities in in vitro translation assays for eIF-4F activity. The phosphorylation of p28 decreases upon heat shock when protein synthesis is repressed. The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF-4F plays a role in the translational control of mRNA binding. Limitations of the in vitro assay system may account for the failure to detect phosphorylation-dependent activity differences.
D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D039562:Eukaryotic Initiation Factor-4F; D006367:HeLa Cells / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007159:Immunologic Tests; D007525:Isoelectric Focusing; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis
D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D010710:Phosphates