Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes.
Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.
Fadden
P
P
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Haystead
T A
TA
Lawrence
J C
JC
D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D007166:Immunosuppressive Agents / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011090:Polyenes / Q000378:metabolism*; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism*; D012694:Serine; D020123:Sirolimus; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D039561:Eukaryotic Initiation Factor-4E; D007166:Immunosuppressive Agents; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011090:Polyenes; D012097:Repressor Proteins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020123:Sirolimus