Abstract
Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.
Highlights
Differential Stimulation of Phosphorylation of Initiation Factors eIF-4F, eIF-4B, eIF-3, and Ribosomal Protein S6 by Insulin and Phorbol Esters*
Stimulation of Phosphorylation of eIF-4F in Quiescent 3T3Ll Cells in Response to Insulin-To examine whether the phosphorylation state of eIF-4F in quiescent, serum-starved 3T3-Ll cells was changed in response to insulin, cells were incubated with [“‘Plorthophosphate in the absence of serum for 1.5 h, and incubated in the absence or presence of 10m7M insulin for 1.5 h. eIF-4F was isolated by m7GTP
The increase in phosphorylation in response to insulin was 2.5-5fold for ~220 and ~25, stimulation as high as lo-fold was seen with the ~220 subunit
Summary
Materials-[‘“PjOrthophosphate was obtained from ICN Radiochemicals; porcine insulin was from Eli Lilly; trypsin (diphenylcarbamyl chloride-treated) and PMA were from Sigma. m7GTP-Sepharose 4B and m’GTP were purchased from Pharmacia LKB Biotechnology Inc. Either insulin in 1 mM HCl or 1 mM HCl alone was added: the cells were incubated further at 37 “C as indicated in the figure legends. For. PMA treatment, cells were incubated with 1 x lo-” M PMA in dimethyl sulfoxide or dimethyl sulfoxide alone [15], at 37 “C, for the times indicated. GTP, 80 mM fl-glycerophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 40 fig/ml each of leupeptin, pepstatin, and antipain), and “P-labeled eIF-4F was isolated immediately by affinity chromatography on m’GTP-Sepharose, as described by Morley and Traugh [15]. Two-dimensional Phosphopeptide Mapping of eIF-QF-Following polyacrylamide gel electrophoresis, individual subunits of eIF-4F were excised from the gel and subjected to trypsin digestion and twodimensional phosphopeptide mapping as described [15, 36].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
