In vivo association of pp60v-src and the gap-junction protein connexin 43 in v-src-transformed fibroblasts.
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 gap-junction protein connexin 43 (Cx43). Previously, we demonstrated that purified, kinase-active pp60src 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 pp60v-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 pp60v-src in v-src-transformed fibroblasts. Cytological localization studies with confocal microscopy demonstrated that pp60v-src and Cx43 were partially co-localized in regions of the plasma membrane. Cx43 and pp60v-src co-immunoprecipitated from v-src-transformed fibroblasts, indicating that the two proteins were associated, and form a stable complex. Furthermore, pp60v-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 pp60v-src phosphorylation. These results provide additional in vivo evidence that Cx43 is a direct substrate of pp60v-src in v-src-transformed fibroblasts. The ability of pp60v-src to alter gap junction-mediated cellular communication may serve as one mechanism by which pp60v-src initiates and/or maintains aspects of cellular transformation.
Basic Science Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 90109-1024, USA.
L W
LW
Loo
M Y
MY
Kanemitsu
A F
AF
Lau
D000818:Animals; D002461:Cell Line, Transformed; D018031:Connexin 43 / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism*; D018613:Microscopy, Confocal; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism* / Q000502:physiology; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats
D018031:Connexin 43; D015688:Oncogene Protein pp60(v-src)