The goal of this research is the functional analysis of pp60, a novel 60 kDa protein substrate of the insulin receptor tyrosine kinase. pp6O rapidly becomes tyrosine phosphorylated in insulin-stimulated cells. This phosphorylation is hypothesized to activate pp60, enabling it to convey the signal from the cell surface insulin receptor to the intracellular metabolic pathways. Recently, we have purified and cloned the cDNA for pp6O. The cloned protein contains several important amino acid structural motifs which mediate protein-protein interactions in diverse signal transduction pathways. We will express the cloned pp6O cDNA as a bacterial fusion protein, which will then be used to generate anti-pp60 antibodies. These antibodies will be used to investigate the parameters of in vivo pp6O phosphorylation in insulin stimulated cells. In addition, using the expressed pp60 fusion protein and anti-pp6O antibodies, we will determine whether pp6O complexes with other endogenous cellular proteins, and investigate the role of specific signalling-protein amino acid structural motifs in mediating these interactions. Analyzing pp6O should improve our understanding of the insulin receptor signalling pathway, and may contribute to the development of improved therapeutics for diabetes mellitus.
