v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication

The mechanism by which v-Src disrupts connexin (Cx)43 intercellular gap jun ctional communication GJC is not clear. In this study, we determined that T yr247 (Y247) and the previously identified Tyr265 (Y265) site of Cx43 were the primary phosphorylation targets for activated Src in vitro. We establis hed an in vivo experimental system by stably expressing v-Src and wild-type (wt) Cx43, of Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout mouse cell line. Wt and mutant Cx43 localized to the plasma membrane in th e absence or presence of v-Src. When coexpressed with v-Src, the Y247F, Y26 5F, and Y247F/Y265F Cx43 mutants exhibited significantly reduced levels of tyrosine phosphorylation compared with wt Cx43, indicating that Y247 and Y2 65 were phosphorylation targets of v-Src in vivo. Most importantly, GJC est ablished by the Y247F, Y265F, and Y247F/Y265F Cx43 mutants was resistant to disruption by v-Src. Furthermore, we did not find evidence for a role for mitogen-activated protein kinase in mediating the disruption of GJC by v-Sr c. We conclude that phosphorylation on Y247 and Y265 of Cx43 is responsible for disrupting GJC in these mammalian cells expressing v-Src.