Connexin 43 interacts with zona occludens-1 and-2 proteins in a cell cycle stage-specific manner

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry ( MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 ( Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 ( ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein "pull down," co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G(0), whereas ZO-2 interaction occurred approximately equally during G(0) and S phases. Since essentially all of the Cx43 in G(0) cells is assembled into Triton X-100-resistant junctions, Cx43ZO-1 interaction may contribute to their stability.