Type I interferons (IFNs) are pleiotropic cytokines that exert multiple biological effects on normal and malignant cells, including antiproliferative, antiviral, and immunomodulatory activities. IFNalpha is used extensively in the treatment of neoplastic diseases and viral infections. Despite the widespread use of IFNalpha in clinical medicine, the specific mechanisms by which this cytokine exerts its effects have not been elucidated. Understanding of such mechanisms will provide valuable information that could be applied in the design of more effective means to use IFNs, and also in the development of novel antineoplastic and antiviral agents. Insulin receptor substrate (IRS)-proteins play a central role in insulin and insulin-like growth factor-1 (IGF-1) signaling by their src homology 2 (SH2)-docking function. These proteins are substrates for the insulin and IGF-1 receptors, and link these receptor tyrosine kinases to downstream signaling-proteins containing SH2 domains. IRS-proteins are also phosphorylated on tyrosine during IFNalpha treatment of cells. The best characterized member of this family, IRS-1, binds the SH2 domains of the p85 regulatory subunit of the phosphatidylinositol 3'-kinase in an IFNalpha-dependent manner, and such an association results in activation of this kinase. The goal of the studies proposed here is to precisely determine the function of IRS-proteins in Type I IFN signaling.
Specific aim A is to determine whether the tyrosine residues of IRS-1 exhibit dual specificity, as tyrosine kinase substrates and as docking sites for IFN-regulated SH2-proteins. This will be achieved by establishing the interactions of IRS-1 with IFNalpha dependent kinases and downstream SH2-proteins, and by mapping the tyrosine sites of IRS-1 that are phosphorylated during IFNalpha stimulation.
Specific aim B is to determine the biological consequences of activation of the IRS-system by IFNs. Studies will be performed to determine the responsiveness of cells that lack expression of IRS proteins to the antiproliferative and antiviral activities of Type 1 IFNs, and the effect of IRS-1 expression on such responses. Other studies will determine the effect of antisense IRS-1 RNA expression on the biological effects of Type I IFNs in IFNalpha-sensitive cells. The possibility that the antiproliferative effect of IFNalpha results in part by an antagonist inhibition of growth factor (insulin/IGF-1) signaling cascades will be also examined. The experiments proposed in specific aim C will determine the IFNalpha- dependent function of the related IRS-2 protein in cells that preferentially express this protein. They will determine whether IRS-2 also functions as a multisite SH2-docking protein, and whether the phosphatidylinositol 3'-kinase pathway is activated by engagement of this protein. Altogether, the proposed studies are aimed towards establishing a model in which signaling specificity for distinct ligands (IFNs, insulin, IGF-1) can be achieved at an early signaling point, through the differential phosphorylation of common docking proteins (IRS-proteins). Competition for the use of these proteins by tumor suppressor cytokines (IFNs) and neoplastic growth factors (insulin/IGF-1) may provide a mechanism by which the proliferative responses of neoplastic cells are regulated.
