c-Src regulates the interaction between connexin-43 and ZO-1 in cardiac myocytes.
Connexin-43 is known to interact directly with ZO-1 in cardiac myocytes, but little is known about the role of ZO-1 in connexin-43 function. In cardiac myocytes, constitutively active c-Src inhibited endogenous interaction between connexin-43 and ZO-1 by binding to connexin-43. In HEK293 cells, by contrast, a connexin-43 mutant lacking the Src phosphorylation site (Tyr265) interacted with ZO-1 despite cotransfection of a constitutively active c-Src. Moreover, in vitro binding assays using recombinant proteins synthesized from regions of connexin-43 and ZO-1 showed that the tyrosine-phosphorylated C terminus of connexin-43 interacts with the c-Src SH2 domain in parallel with the loss of its interaction with ZO-1. Cell surface biotinylation revealed that, by phosphorylating Tyr265, constitutively active c-Src reduces total and cell surface connexin-43 down to the levels seen in cells expressing a mutant connexin-43 lacking the ZO-1 binding domain. Finally, electrophysiological analysis showed that both the tyrosine phosphorylation site and the ZO-1-binding domain of connexin-43 were involved in the regulation of gap junctional function. We therefore conclude that c-Src regulates the interaction between connexin-43 and ZO-1 through tyrosine phosphorylation and through the binding of its SH2 domain to connexin-43.
Toyofuku
T
T
Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. toyofuku@mr-path.med.osaka-u.ac.jp
Akamatsu
Y
Y
Zhang
H
H
Kuzuya
T
T
Tada
M
M
Hori
M
M
D002460:Cell Line; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D018031:Connexin 43; D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src)