A central aspect of the macrophage response to bacterial lipopolysaccharide (LPS) is the coordinated synthesis and release of proinflammatory cytokines, including TNF-alpha and IL-12. IL-12, in particular, is crucial for the induction of IFN-gamma secretion, and the development of a Th1 response. Activation of macrophage FcgammaR with immune complexes inhibits the LSP-stimulated synthesis of IL-12, but not of TNF-alpha. We have found that this phenotype is mimicked by dominant negative (dn) constructs of a novel microtubule associated serine/threonine kinase, MAST2O5, which we identified by expression cloning for inositol hexakisphosphate (IP6) binding proteins. Exogenous IP6 and FcgammaR activation inhibit macrophage IL-12 synthesis, and target MAST2O5 for proteosomal degradation. MAST2O5 is thus pivotally placed in the FcgammaR-regulated IL-12 response pathway. However, little is known about MAST2O5, the pathways and proteins with which it interacts, or its substrates. This proposal is structured around three aims: 1) We will analyze the signaling pathways affected by MAST205. First, we will focus on NF-kappaB, because we have shown that LPS-activated NF-kappaB promoter activity is inhibited 90% by dn-MAST205 We will also examine other aspects of the macrophage response to FcgammaR activation modulated by MAST2O5; 2) We will determine, by immunoprecipitation and mass spectrometry, the proteins that interact with MAST2O5, how the interactions vary with different physiological stimuli, and post-translational modifications of MAST2O5; 3) We will analyze the effects of different kinase and phosphatase inhibitors on IL-12 synthesis and MAST2O5 stability, and screen peptide phage libraries for MAST2O5-binding peptides that inhibit MAST2O5 autophosphorylation and/or macrophage NF-gammaB activation by LPS. These studies will advance our understanding of IL-12 regulation, which may aid in control of the inflammatory response.
