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In vivo association of pp60 v‐src and the gap‐junction protein connexin 43 in v‐ src –transformed fibroblasts
Author(s) -
Loo Lenora W. M.,
Kanemitsu Martha Y.,
Lau Alan F.
Publication year - 1999
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/(sici)1098-2744(199907)25:3<187::aid-mc5>3.0.co;2-o
Subject(s) - proto oncogene tyrosine protein kinase src , biology , phosphorylation , connexin , tyrosine phosphorylation , microbiology and biotechnology , gap junction , sh2 domain , tyrosine kinase , kinase , signal transduction , intracellular
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 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 co‐localized 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. Mol. Carcinog. 25:187–195, 1999. © 1999 Wiley‐Liss, Inc.