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 G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.
Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Deepika
D
Singh
Joell L
JL
Solan
Steven M
SM
Taffet
Ronald
R
Javier
Paul D
PD
Lampe
D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology; D017629:Gap Junctions; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016196:S Phase; D014357:Trypsin / Q000737:chemistry; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D018031:Connexin 43; D003902:Detergents; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D017830:Octoxynol; D005982:Glutathione Transferase; D014357:Trypsin