Activation of the Phosphatidylinositol 3-Kinase Serine Kinase by IFN-α

During engagement of the type I IFN receptor, IRS-1 is phosphorylated on tyrosine and associates with the p85 regulatory subunit of the phosphatidylinositol (PI) 3′-kinase, which is a dual-specificity enzyme possessing both lipid and serine kinase activities. We sought to determine whether treatment of cells with IFN-α activates the PI 3′-kinase serine kinase. ³²P-labeling experiments and phosphoaminoacid analysis of immunoprecipitated IRS-1 protein demonstrated that, in addition to tyrosine phosphorylation, IFN-α induces its phosphorylation on serine residues. In vitro kinase assays on αIRS-1 immunoprecipitates also demonstrated IFN-α-dependent serine phosphorylation of IRS-1, suggesting that the protein associates with an IFN-a-regulated serine kinase. Furthermore, IFN-α-dependent phosphorylation of IRS-1 was detected in in vitro kinase assays on αp85 immunoprecipitates, and was inhibited by pretreatment of cells with the specific PI 3′-kinase inhibitor wortmannin, consistent with a regulatory role of the PI 3′-kinase serine kinase on the phosphorylation of the protein. Treatment of cells with wortmannin also inhibited the phosphorylation of the p85 subunit of PI 3′-kinase and the type I IFN-regulated activation of the Map kinase, but had no inhibitory effect on the IFN-α-induced activation of Tyk-2 and Jak-1 kinases nor on the activation of Stat-1, Stat-2, and Stat-3. Taken all together, these data establish that the PI 3′-kinase serine kinase is activated by IFN-α and may play an important role in the transmission of type I IFN receptor-generated signals.