Phosphorylation Controls the Interaction of the Connexin43 C-Terminal Domain with Tubulin and Microtubules

Abstract

Connexins are structurally related transmembrane proteins that assemble to form gap junction channels involved in the mediation of intercellular communication. It has been shown that the intracellular tail of connexin43 (Cx43) interacts with tubulin and microtubules with putative impacts on its own intracellular trafficking, its activity in channel communication, and its interference with specific growth factor signal transduction cascades. We demonstrate here that the microtubule binding of Cx43 is mainly driven by a short region of 26 amino acid residues located within the intracellular tail of Cx43. The nuclear magnetic resonance structural analysis of a peptide (K26D) corresponding to this region shows that this peptide is unstructured when free in solution and adopts a helix conformation upon binding with tubulin. In addition, the resulting K26D-tubulin molecular complex defines a new structural organization that could be shared by other microtubule partners. Interestingly, the K26D-tubulin interaction is prevented by the phosphorylation of K26D at a src kinase specific site. Altogether, the results elucidate the mechanism of the interaction of Cx43 with the microtubule cytoskeleton and propose a pathway for understanding the microtubule-dependent regulation of Cx43 gap junctional communications and the involvement of Cx43 in TGF-β signal transduction.

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

“…The interaction between Cx43 and tubulins occurs in a 26 amino acid region (in the region of aa 228-262) in the Cx43 C terminus, which results in the formation of helical regions at the point of interaction between the two proteins. This process was recently demonstrated to be regulated through v-src phosphorylation at the Tyr247 of the Cx43 C terminus (Giepmans et al, 2001b;Saidi Brikci-Nigassa et al, 2012). The interaction between connexins and tubulins regulates membrane targeting, localization within the plasma membrane, gap junction plaque formation, and TGF-b signaling (Shaw et al, 2007;Saidi BrikciNigassa et al, 2012).…”

“…Microdomains Regulating the Intracellular Trafficking of Connexins. Once formed, vascular connexin hemichannels are actively transported to the plasma membrane through direct binding of their C-terminal and intracellular loop regions with proteins such as tubulin (a and b) (Giepmans et al, 2001b;Thomas et al, 2005;Kang et al, 2009;Saidi Brikci-Nigassa et al, 2012). Associations between Cx43 and tubulins (a and b) have been shown through coimmunoprecipitation, immunofluorescence overlap, analytical size exclusion chromatography, nuclear magnetic resonance, pulldown assays, and site-directed mutagenesis in a variety of studies (e.g., Giepmans et al, 2001b;Saidi BrikciNigassa et al, 2012).…”

“…Kinases and sites within Cx43 have been well characterized, including phosphorylation by kinases from the src family of Tyr265, and phosphorylated by MAPK of Ser255/(Ser262)/Ser279/ Ser282 residues (Solan and Lampe, 2009;Johnstone et al, 2012a;Chen et al, 2013). Presumptively, these posttranslational modifications induce structural changes within the connexin C-terminal region that allow for further protein interactions with this region (Saidi Brikci-Nigassa et al, 2012). However, given the lack of data on the effect of posttranslational modifications on the structure of connexin C terminus, the exact consequences of these modifications remain unclear.…”

Effects of Phosphorylation on the Structure and Backbone Dynamics of the Intrinsically Disordered Connexin43 C-terminal Domain

“…Evidence has emerged that a phosphate can directly affect binding of the Cx43CT domain with molecular partners by blocking the binding site (e.g. ZO-1 (27) and tubulin (28)) or by being directly involved in the interaction (e.g. Nedd4 (29,30)).…”

Tyrosine phosphatase TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

“…Second, phosphorylation can directly (and possibly indirectly, see Grosely et al, 2013) alter the binding affinities of proteins involved in Cx43 regulation. For example, the aromatic ring of Y247 is directly involved in microtubule binding, and Y247 phosphorylation inhibits this interaction (Saidi Brikci-Nigassa et al, 2012). Because microtubules play important roles in rapid connexin delivery to gap junction plaques (Shaw et al, 2007), pY247 would be detrimental to Cx43 intercellular communication.…”