title: Control of PHAS-I by insulin in 3T3-L1 adipocytes. Synthesis, degradation, and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-independent pathway.
abstract: PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-I eukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H6]PHAS-I when the [H6]PHAS-I.eIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-I.eIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70S6K. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.
authors.affiliation: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Lin
authors.forename: X
authors.initials: X
authors.lastname: Kong
authors.forename: A R
authors.initials: AR
authors.lastname: Saltiel
authors.forename: P J
authors.initials: PJ
authors.lastname: Blackshear
authors.forename: J C
authors.initials: JC
authors.lastname: Lawrence
mesh_terms: D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D039642:Eukaryotic Initiation Factors; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis; D009124:Muscle Proteins; D010750:Phosphoproteins / Q000096:biosynthesis*; D010766:Phosphorylation / Q000187:drug effects; D011090:Polyenes / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020123:Sirolimus
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D039642:Eukaryotic Initiation Factors; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D011090:Polyenes; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C495693:Slc2a4 protein, mouse; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020123:Sirolimus