Phosphorylation of Gap Junction Proteins

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DESCRIPTION: (provided by applicant) Gap junctional communication is critically important in many cell processes including control of cell proliferation, embryonic development, and cell differentiation. These pathways provide for cell-to-cell diffusion of small molecules (<1000 Da) including ions, metabolites, and second messengers. In vertebrates, gap junctions are composed of proteins from the connexin family, which contains over a dozen members. Channels composed of different connexins can be opened or closed to varying degrees by changes in transjunctional voltage, pH, and phosphorylation. This proposal is focused on connexins 43 (Cx43) and 45 for several reasons; both are known to be phosphorylated on multiple serines, both are positively/negatively regulated by kinase effectors such as TPA, however, they have very different conducting properties and share little sequence homology in the cytoplasmic tail region aside from a series of double serine repeats. This proposal examines the role of phosphorylation in the regulation of these gap junction proteins and the channels that they form. We hypothesize that in addition to effects on gating, phosphorylation is important for the regulation of other aspects of Cx43's "life cycle" (i.e., connexin oligomerization, connexin trafficking, gap junction assembly, and protein turnover). Our specific aims are to (1) determine the phosphorylation events within Cx43 and Cx45, which regulate connexin trafficking, gap junction assembly, gating and turnover in homeostatic cells. (2) Characterize kinases that bind to and regulate connexin function. (3) Determine the cell cycle dependent changes in connexin phosphorylation that allow connexin redistribution during mitosis. We plan to apply our knowledge of connexin phosphorylation and phosphorylation specific probes to the analysis of connexin status in wounded skin and the remodeling of gap junctions during heart disease.