Connexin 43 Phosphorylation as a Regulatory Mechanism of Src Activity

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PROJECT SUMMARY Connexin 43 (Cx43) is a protein constituent of vertebrate gap junctions (GJs) - cellular structures key to direct cell-cell communication. Cx43-based GJs are known to be regulated through phosphorylation by several kinases on the cytoplasmic C-terminal tail (CT) of Cx43. Recent studies by the PI and others have indicated that Cx43-CT functions as a cell-signaling hub that interacts with and regulates a large number of kinases, phosphatases, scaffolding proteins, and cytoskeletal components. One of the kinases that interacts with and phosphorylates Cx43 is the proto-oncogenic tyrosine kinase, Src. Interestingly, ectopic expression of Cx43 in cancer cells with overactive Src leads to inhibition of Src activity and a decrease in cellular proliferation. While it is known that Src inhibition is due to its binding to Cx43-CT, along with the recruitment of two Src inhibitors (the phosphatase PTEN and C-terminal Src kinase, Csk), the precise molecular mechanisms that regulate these processes are not understood. We hypothesize that Cx43’s ability to recruit and inhibit Src is driven and regulated by a series of specific and sequential phosphorylation events taking place throughout Cx43-CT. Here, we propose three interconnected, but non-overlapping, specific aims, with the ultimate goals of (1) determining the specific sequence of phosphorylation events on Cx43 that regulate Cx43/Src interaction, and (2) developing Cx43-based peptides for use as Src inhibitors in cancer cells. In the first two aims, we will identify and interrogate particular phosphorylation sites as toggle switches that regulate the interactions between Cx43 and binding partners (e.g., Src, PTEN, Csk), with subsequent effects on signaling pathways and cell phenotypes. In the third aim, we will develop Cx43-based peptides and test their ability to modulate the Cx43/Src interaction and Src activity in living cells. To achieve these goals, we will combine expertise in protein biochemistry, kinase-substrate interactions (both in vitro and in cells), phosphoproteomics and mass spectrometry analyses of complex sample mixtures, and specific delivery of agents to cancer cells. These studies will substantially advance our molecular understanding of Src-mediated cell signaling, with a focus on the specific role of Cx43 as an interacting hub. It will also allow us to delineate multiple signaling pathways in both healthy and cancer cells. The Cx43-based peptides that we propose to develop will represent a novel way not only to tease out Cx43/Src regulatory mechanisms in live cells but also to target the aberrant activity of Src in cancers. We expect that the basic framework developed here for Cx43 can be extended to other connexins and to the examination of other signaling pathways. Finally, these studies will continue to provide invaluable research experience and professional development for many undergraduate Life Science majors at Moravian University.