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, connexin 43 (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. 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. 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 paper demonstrates that kinase active pp60c-src 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.
Highlights
Posed of four transmembrane domains, two extracellular loops, an intracellular loop, and amino- and carboxyl-terminal domains located in the cytoplasm [9,10,11]
Gap junctional communication (GJC)1 has been implicated in the regulation of growth control based on experiments linking the permeability of the gap junction channel with cellular transformation
These results suggested that pp60v-src may directly regulate channel gating via tyrosine phosphorylation of connexin 43 (Cx43) on residue 265, it is possible that these events in the oocytes do not completely reflect the events in mammalian cells
Summary
Molecular Biologyand VirologyLaboratory, Salk Institute, La Jolla, CA 92138. II To whom correspondence should be addressed. Cells grown at the nonpermissive temperature, with normal levels of GJC contained Cx43 phosphorylated only on phosphoserine (Ser(P)) residues These results indicated that may decrease by inducing the phosphorylation of Cx43 on tyrosine. Oocytes coexpressing a mutated form of Cx43 (tyrosine 265 mutated to phenylalanine) and pp60v -src did not demonstrate a decrease in GJC or tyrosine phosphorylation on Cx43 [41] These results suggested that pp60v-src may directly regulate channel gating via tyrosine phosphorylation of Cx43 on residue 265, it is possible that these events in the oocytes do not completely reflect the events in mammalian cells. The results from this study indicated that Cx43 can serve as a substrate of activated pp60Src in in vitro kinase reactions and in intact mammalian and insect cells These data strongly suggest that the tyrosine residue(s) phosphorylated by pp60src are located in the carboxyl tail region of Cx43. Since tyrosine phosphorylation on Cx43 has been correlated with a down-regulation in GJC, these results suggest that activated pp60src has a direct role in modulating the gating of gap junctions
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