STIMULATION OF PROTEIN-SYNTHESIS, EUKARYOTIC TRANSLATION INITIATION-FACTOR 4E PHOSPHORYLATION, AND PHAS-I PHOSPHORYLATION BY INSULIN REQUIRES INSULIN-RECEPTOR SUBSTRATE-1 AND PHOSPHATIDYLINOSITOL 3-KINASE

Insulin rapidly stimulates protein synthesis in a wide variety of tiss ues. This stimulation is associated with phosphorylation of several tr anslational initiation and elongation factors, but little is known abo ut the signaling pathways leading to these events. To study these path ways, we have used a myeloid progenitor cell line (32D) which is depen dent on interleukin 3 but insensitive to insulin because of the very l ow levels of insulin receptor (IR) and the complete lack of insulin re ceptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expressio n of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stim ulation of the 70-kDa S6 kinase (pp70(S6K)) by the endogenous LR. Howe ver, expression of both IR and IRS-1 is required for stimulation of pr otein synthesis. Moreover, this effect requires activation of phosphat idylinositol 3-kinase (P13K), as determined by wortmannin inhibition a nd the use of an IRS-1 variant lacking all Tyr residues except those w hich activate P13K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21(ras) or SH-PTP2, since IR S-1 variants lacking the; SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70(S6K), since rapamycin, whil e strongly inhibiting the synthesis of a small subset of growth-regula ted proteins, only slightly inhibits total protein synthesis. Recruitm ent of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhib ition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-l-mediated stimulation of P13K and pp70(S6 K) but not mitogen-activated protein kinase or SH-PTP2.