BIOMOLECULAR INTERACTION ANALYSIS OF IFN-GAMMA-INDUCED SIGNALING EVENTS IN WHOLE-CELL LYSATES - PREVALENCE OF LATENT STAT1 IN HIGH-MOLECULAR-WEIGHT COMPLEXES

The basic framework for the JAK/STAT pathway is well documented. Recru itment of latent cytoplasmic STAT transcription factors to tyrosine ph osphorylated docking sites on cytokine receptors and their JAK-mediate d phosphorylation instigates their translocation to the nucleus and th eir ability to bind DNA, The biochemical processes underlying recruitm ent and activation of this pathway have commonly been studied in recon stituted in vitro systems using previously defined recombinant signali ng components. We have dissected the Interferon gamma (IFN gamma) sign al transduction pathway in crude extracts from wild-type and STAT1-neg ative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in de tergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containin g peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at le ast for IFN gamma-primed cells, is available for recruitment to the ac tivated IFN gamma-receptor from only a subset of such complexes; (3) a ctivated STAT1 is released from the receptor as a monomer.