Phosphoinositide 3-kinases are critical regulators of proliferation, motility, apoptosis and vesicular trafficking, whose activity and/or expression is elevated in human cancers. This proposal focuses on the Class IA (p85/p 110) PT 3-kinases, which are regulated by receptor tyrosine kinases, and in intact cells produce the second messenger PI about3,4,5]P3. These enzymes contain separate regulatory (p85) and catalytic (p110) domains. We have previously shown that p85/p110 dimers are activated when phosphotyrosine peptides bind to the SH2 domain of p85. We have also established that the p85 regulatory subunit is an inhibitor of the p110 catalytic subunit, and that activation of the p85/p110 heterodimer reflects disinhibition of p110. The minimal fragment of p85 required for regulation of p110 is a putative coiled-coil region, the iSH2 (inter-SH2) domain, linked at its amino terminus to a single SH2 domain (the nSH2 domain). Isolated nSH2 domains do not bind p110 or affect its activity. Isolated iSH2 domains are sufficient to bind to the N-terminus of p110 (residues 1-108), but this binding does not affect p110 activity. Inhibition is only seen with the nSH2-iSH2 fragment (hereafter referred to as nSH2-iSH2). Thus, the presence of the nSH2 domain modulates the effect of iSH2 binding on p110 activity. The first three specific aims examine the mechanism of p110 regulation by nSH2-iSH2.
Aim 1 will use EPR spectroscopy and LC/MS analysis of deuterium exchange to test the predicted structure of nSH2-iSH2 and identify regions that undergo conformational changes upon phosphopeptide binding.
Aim II will introduce tethered chemical proteases at specific sites in nSH2-iSH2, in order to map its sites of contact with p110.
Aim III will use NMR spectroscopy to solve the structure of nSH2-iSH2 and the p85-binding domain of p110. Finally, Aim IV is motivated by our finding that in a rat adenocarcinoma cell line, one isoform of the p110 catalytic subunit (p1lOa) is required for EGF-stimulated cytoskeletal responses, whereas the other isoform (p1lOB) is not. We hypothesize that p110a and p110B activate different sets of downstream effectors, which couple differently to the cytoskeleton; we will identify the downstream components that are differentially activated by p1lOa and p1lOB. Successful completion of these experiments will substantially advance our understanding of signal transduction by this critical and physiologically important signaling enzyme.
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