ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

Abstract

We have previously reported that protein kinase C gamma (PKCγ) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKCγ translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKCγ-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKCγ with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKCγ, the Cx43 did not disassemble out of plaques even though the PKCγ enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKCγ-driven disassembly response.

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…Phosphorylation of Cx43 at the Ser368 residue is known to decrease gap junction communication and is associated to internalization of the protein; in other words, it is associated with reduced plaque stability. This phosphorylation is reportedly dependent on Cx43 interaction with ZO-1, because in the absence of ZO-1, Cx43 can interact with but cannot be phosphorylated by PKC« and efficiency of disassembly of gap junctions is reduced (Akoyev and Takemoto, 2007). These results suggest that ZO-1 interactions cause structural changes in the Cx43-CT that allow for PKC phosphorylation (Akoyev and Takemoto, 2007;O'Quinn et al, 2011).…”

“…This phosphorylation is reportedly dependent on Cx43 interaction with ZO-1, because in the absence of ZO-1, Cx43 can interact with but cannot be phosphorylated by PKC« and efficiency of disassembly of gap junctions is reduced (Akoyev and Takemoto, 2007). These results suggest that ZO-1 interactions cause structural changes in the Cx43-CT that allow for PKC phosphorylation (Akoyev and Takemoto, 2007;O'Quinn et al, 2011). Thus, although ZO-1 interaction with Cx43 may not be critical in the formation of gap junction channels, ZO-1 may act to regulate their function through membrane targeting and molecular inhibition by limiting gap junction formation and promoting endocytosis from the gap junction plaque.…”

The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

“…It has been variously proposed that ZO-1 enhances GJ assembly (Laing et al 2005), plays a role in GJ disassembly (Akoyev and Takemoto 2007), or mediates GJ internalization (Barker et al 2002;Segretain et al 2004). ZO-1 also has been implicated in the regulation of intercellular communication through Cx43 GJs (Akoyev and Takemoto 2007;Girao and Pereira 2007;Laing et al 2005;Toyofuku et al 2001;van Zeijl et al 2007). Given the predominant edge-localization of ZO-1, it is possible that many of these processes are integrated at plaque edges, with modality determined by which molecules ZO-1 actively links to (or disengages from) Cx43.…”

Investigation of the Reciprocal Relationship between the Expression of Two Gap Junction Connexin Proteins, Connexin46 and Connexin43

“…Western Blot and Antisera-Western blot analyses were performed as previously described (24,25,28). Mouse anti-N-terminal-Cx43 (anti-Cx43 NT) was purchased from Fred Hutchinson Cancer Center (Seattle, WA); rabbit anti-phosphoCx43 (Ser-368) was from Cell Signaling Technologies (Danvers, MA); rabbit anti-C-terminal-Cx43 (anti-Cx43 CT, 1:5000) and mouse anti-␤-actin (1:10000) were from Sigma-Aldrich; rabbit anti-Cx46 (1:2000) was from U.S. Biologicals (Swampscott, MA); mouse anti-Cx50 (1:4000) was from Zymed Laboratories Inc.-Invitrogen (San Franscisco, CA); and mouse anti-EGFP antibody (1:5000) was purchased from Clontech.…”