In vivo association of pp60v-src and the gap-junction protein connexin 43 in v-src–transformed fibroblasts

✍ Scribed by Lenora W. M. Loo; Martha Y. Kanemitsu; Alan F. Lau

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 194 KB
• Volume: 25
• Category: Article
• ISSN: 0899-1987
• DOI: 10.1002/(sici)1098-2744(199907)25:3<187::aid-mc5>3.0.co;2-o

✦ Synopsis

v-src±transformed fibroblasts have significantly reduced levels of gap junction±mediated intercellular communication. This observed downregulation of cellular communication has been associated with tyrosine phosphorylation of the gapjunction protein connexin 43 (Cx43). Previously, we demonstrated that purified, kinase-active pp60 src phosphorylates Cx43 in vitro (J Biol Chem 1995; 270:12751±12761). More recently, we reported that this association is mediated by the SH2 and SH3 domains of pp60 v-src (J Biol Chem 1997;272:22824±22831). In this report, we present in vivo evidence supporting the hypothesis that Cx43 is an endogenous substrate of pp60 v-src in v-src±transformed fibroblasts. Cytological localization studies with confocal microscopy demonstrated that pp60 v-src and Cx43 were partially colocalized in regions of the plasma membrane. Cx43 and pp60 v-src co-immunoprecipitated from v-src±transformed fibroblasts, indicating that the two proteins were associated, and form a stable complex. Furthermore, pp60 v-src could phosphorylate co-immunoprecipitated Cx43 in an immune-complex kinase assay. Two-dimensional phosphopeptide mapping of the immune-complexed Cx43 phosphorylated in vitro demonstrated that the sites of tyrosine phosphorylation were consistent with previously identified sites of pp60 v-src phosphorylation. These results provide additional in vivo evidence that Cx43 is a direct substrate of pp60 v-src in v-src±transformed fibroblasts. The ability of pp60 v-src to alter gap junction±mediated cellular communication may serve as one mechanism by which pp60 v-src initiates and/or maintains aspects of cellular transformation.