pp60src-mediated phosphorylation of connexin43, a gap junction protein

Abstract

Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease In gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Confocal microscopy data indicates that the two proteins are within physical proximity allowing for a potential kinase-substrate interaction. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43 we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially-purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60src. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. This phosphorylation event occurred in the COOH-tail region of the Cx43 molecule because pp60src phosphorylated a GST-fusion protein containing the COOH-tail region of Cx43 and the phosphotryptic peptides generated using this construct comigrated with the subset mentioned above. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60v-src and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this dissertation demonstrates that kinase active pp60src is capable of phosphorylating Cx43 In a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down regulation of gap-junctional communication, these results suggest that pp60v-src regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.