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 Ser(64), the preferred site of MAP kinase, Binding of the Ala(64) mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating th at phosphorylation of sites other than Ser(64) control binding. To ide ntify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma -P-32]ATP and then cleaved proteolytically before the resulting phosph opeptides were isolated by reverse phase chromatography and directly i dentified by amino acid sequencing, Phosphorylated residues were locat ed by determining the cycles in which P-32 was released when phosphope ptides were subjected to sequential Edman degradation. With an extende d incubation in vitro, MAP kinase phosphorylated Thr(36), Thr(45), Ser (64), Thr(69), and Ser(82). In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin alt hough there were differences in the magnitude of the effects, A form o f PHAS-I phosphorylated exclusively in Thr(36) remained bound to eIF-4 E, indicating that phosphorylation of Thr(36) is insufficient for diss ociation of the PHASI.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PH AS-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-d irected protein kinase.