Insulin and phorbol ester stimulate initiation factor eIF-4E phosphorylation by distinct pathways in Chinese hamster ovary cells overexpressing the insulin receptor.
We have developed a one-dimensional isoelectric focusing technique to measure changes in the steady-state phosphorylation of the cap-binding initiation factor, eIF-4E. We have used a Chinese hamster ovary cell line transfected with the human insulin receptor (CHO.T cells) to study the regulation of eIF-4E phosphorylation by insulin and other stimuli. Exposure of CHO.T cells to insulin, phorbol ester or serum resulted in a rapid increase (up to twofold) in eIF-4E phosphorylation. As a control, we have also performed experiments with the parental cell line, CHO.K1 cells, in which both serum and phorbol ester, but not nanomolar concentrations of insulin, produce similar changes in eIF-4E phosphorylation. We have used two complementary approaches to study the role of protein kinase C (PKC) in these responses: a highly specific inhibitor of PKC and down-regulation of PKC by prior treatment of the cells with phorbol ester. In CHO.T cells, both approaches indicate that PKC is required for the response to phorbol ester but that insulin and serum each increase eIF-4E phosphorylation by a mechanism(s) independent of this protein kinase. Similarly, PKC is necessary for the effects of phorbol ester, but not of serum, on eIF-4E phosphorylation in CHO.K1 cells. These data indicate that multiple signal transduction mechanisms are involved in the modulation of eIF-4E phosphorylation and the implications of these findings are discussed.
Flynn
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A
Department of Biosciences, University of Kent at Canterbury, United Kingdom.
Proud
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CG
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D039561:Eukaryotic Initiation Factor-4E; D007328:Insulin; D010448:Peptide Initiation Factors; D011972:Receptor, Insulin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate