The src oncogene of the Rous sarcoma virus encodes, pp60v-src, the prototypical cytoplasmic tyrosine protein kinase. The epidermal growth factor receptor (EGFR) is also a tyrosine protein kinase, but of the transmembrane receptor class. These tyrosine kinases have as a common cellular target, connexin43, which comprises aqueous plasma membrane channels interconnecting the cytoplasms of adjacent cells in tissues or in culture. The passage of ions and other small molecules through gap junctions is known to be important for the proper functioning of numerous normal tissues. It has been postulated that loss of gap-junctional communication (gjc) produces a concomitant interruption of growth regulatory signals, passing through the channels, which may result in the uncontrolled cell growth associated with tumor formation. Development of invasive and metastatic tumors in vivo could possibly result from the lack of this type of intercellular communication. The pp60v-src and EGFR tyrosine kinases induce phosphorylation of connexin43 on tyrosine and serine residues, respectively, and profoundly disrupt gjc. The overall objective of this research proposal is to obtain a more complete and detailed understanding of the phosphorylation mechanisms by which pp60v- src and the EGFR modulate the biological activity of their common target, connexin43. This effort will also clarify the role that gap junctions play in v-src-induced cellular transformation by discerning which transformed cell properties are dependent upon the disruption of connexin43 function.
Specific Aim 1 will elucidate the molecular mechanisms involved in the pp60v-src-induced tyrosine phosphorylation of connexin43 and alteration of its activity through identification of connexin43 phosphotyrosine sites, biochemical and biological analysis of prepared phosphotyrosine site-directed mutants, and determination if connexin43 is a direct substrate of pp60v-src by the use of in vitro kinase assays and connexin43 channels reconstituted in lipid bilayers.
Specific Aim 2 will define the role of pp60v-src-stimulated phosphoserine sites in connexin43 through the biochemical and biological analysis of connexin43 site-directed mutants and the study of pp60v-src-activated serine kinase.
Specific Aim 3 will examine the participation of EGFR- activated signalling serine kinases in alterations of connexin43 phosphorylation and gjc by determining if MAP kinase, the Shc signaling pathway, or other activated serine kinases are involved in these effects.
Specific Aim 4 will determine if certain phenotypic properties of pp60v- src-initiated neoplastic cellular transformation are dependent upon disruption of gjc by studying: i) v-src-transformed cells whose ability to communicate through gap junctions is restored by transfection and expression of connexin32 and ii) the ability of pp60v-src to transform fibroblasts which lack connexin43 due to expression of connexin43 antisense DNA or knockout of its gene.
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