Control of PHAS-I phosphorylation in 3T3-L1 adipocytes: effects of inhibiting protein phosphatases and the p70S6K signalling pathway.
PHAS-I is a recently discovered regulator of translation initiation. Non-phosphorylated PHAS-I binds and inhibits eukaryotic initiation factor-4E, the mRNA cap-binding protein that mediates a rate-limiting step in translation initiation. When PHAS-I is phosphorylated in response to insulin, the PHAS-I/eukaryotic initiation factor-4E complex dissociates. The present study was conducted to investigate mechanisms involved in the control of PHAS-I. Phosphorylation of PHAS-I was monitored by immunoblotting after subjecting extracts to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This was possible because phosphorylation markedly decreases the electrophoretic mobility of PHAS-I. Incubating 3T3-L1 adipocytes with rapamycin and wortmannin inhibited insulin-stimulated phosphorylation of PHAS-I at concentrations similar to those that inhibited activation of p70S6K. Both agents increased the amount of PHAS-I that co-purified with eukaryotic initiation factor-4E when extracts were fractionated using a cap affinity resin, indicating that PHAS-I binding to the initiation factor was increased. Incubating adipocytes with the protein phosphatase inhibitors, calyculin A and okadaic acid, increased PHAS-I phosphorylation and opposed the effects of rapamycin on decreasing PHAS-I phosphorylation. However, neither okadaic acid nor calyculin A abolished the effects of rapamycin on PHAS-I. These results suggest that the phosphorylation of PHAS-I in response to insulin occurs via the p70S6K signalling pathway. By regulating eukaryotic initiation factor-4E, PHAS-I may have important roles in the control of both protein synthesis and mitogenesis.
Lin
T A
TA
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri, USA.
Lawrence
J C
JC
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D005347:Fibroblasts; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D008387:Marine Toxins; D051379:Mice; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000187:drug effects / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology; D011817:Rabbits; D012097:Repressor Proteins / Q000187:drug effects / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases / Q000187:drug effects / Q000378:metabolism*; D020123:Sirolimus; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D007106:Immune Sera; D007328:Insulin; D008387:Marine Toxins; D010080:Oxazoles; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011090:Polyenes; D012097:Repressor Proteins; D019319:Okadaic Acid; C059041:calyculin A; D019893:Ribosomal Protein S6 Kinases; D010749:Phosphoprotein Phosphatases; D020123:Sirolimus; D000077191:Wortmannin