CONNEXIN 43 REGULATION IN PROLIFERATIVE VASCULAR DISEASE

University Of Hawaii At Manoa, Honolulu HI

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Abstract

DESCRIPTION (provided by applicant): The overall goal of these studies is to understand how the regulation of intercellular gap junctional communication (GJC) contributes to the regulation of cell growth. We will specifically examine the regulation of the connexin43 (Cx43) gap junction protein by mitogen-induced phosphorylation and novel CA3 -interacting proteins. CA3 has been termed a "tumor suppressor" due to its association with decreased rates of cell growth, a property thought to be mediated by the intercellular exchange of growth regulatory signals through gap junctions. Stimulation by growth factors or expression of viral oncogenes in normal cells is associated with increased CA3 phosphorylation and altered GJC. The v-Src oncoprotein directly phosphorylates CA3 on tyrosine, whereas, activation of the epidermal growth factor receptor initiates activation of the mitogen-activated protein kinase (MAPK) signaling cascade and MAPK-mediated serine phosphorylation on CA3. These phosphorylation events are associated with disruption of GJC. In these studies we will examine the regulation of CA3 by activation of G protein-coupled receptor signaling pathways. We will utilize specific reagents and cell lines stabily expressing CA3 mutants to identify signaling events that mediate CA3 phosphorylation and determine whether phosphorylation disrupts GJC. Increased phosphorylation will be demonstrated by an increase in the phosphorylated isoforms of CA3 in radiolabeled cells. We will determine whether phosphorylation occurs on serine or on tyrosine by phosphoamino acid analysis and measure GJC by the transfer of fluorescent dye from a micro injected cell to neighboring cells through gap junctions. We will also investigate the functional significance of the interaction of CA3 with two proteins identified in a yeast two-hybrid screen: 14-3-3 theta and CIP 77 (a protein with homology to the TOG (tumor over-expressing gene) protein). Interacting proteins may be involved in regulating Cx43 expression, post-translational modification, protein turnover, gap junction assembly, or the trafficking of CA3 to and from the membrane. Understanding how mitogen-induced phosphorylation and interacting proteins regulate CA3 may shed light on the growth suppressive function of Cx43 and on how the disruption of GJC contributes to increased cell growth in diseases such as vascular stenosis and cancer. The activation of G protein-coupled receptor signaling and the activation of MAPK, and cSrc have been demonstrated in vascular restenosis. Thus, understanding the regulation of CA3 by mitogen-induce phosphorylation and by interacting proteins is expected to provide insight into mechanisms contributing to the disregulation of growth that is observed in hyperproliferative diseases such as vascular restenosis.