Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2

Abstract

Gap junctions are integral membrane proteins that enable the direct cytoplasmic exchange of ions and low molecular weight metabolites (Ͻ1 kDa) between adjacent cells. They provide an intercellular pathway for the propagation and/or amplification of signal transduction cascades triggered by cytokines, growth factors, and other cell-signaling molecules involved in growth regulation and development. Gap junctions are formed by the apposition of connexons from adjacent cells, where each connexon is formed by six connexin proteins. Connexins are tetraspanning transmembrane proteins with intracellular N and C termini (CT). 2 There are 21 human isoforms, and although there is significant sequence homology among connexins, their major divergence occurs in the cytoplasmic domains. Connexin43 (Cx43), the most ubiquitously expressed connexin isoform, has been well studied in terms of structure, function, and regulation (1).Cx43 channels can be regulated by a variety of molecules and physiological conditions (e.g. Ca 2ϩ , pH, and intercellular voltage), including through phosphorylation (2, 3). All stages of the Cx43 life cycle (i.e. trafficking, assembly/disassembly, degradation, and channel gating (3)), as well as the regulation of electrical and metabolic coupling and of interactions with other proteins, involve phosphorylation. The Cx43CT is differentially phosphorylated on at least 20 residues, and significant progress has been achieved in characterizing the kinases involved (2, 4 -6). Unfortunately, an understanding of the mechanism(s) by which Cx43 phosphorylation alters channel function is lacking. Several problems contributing to this difficulty include the transient nature of a particular CT phosphorylation state, the ability of many kinases to phosphorylate more than one CT residue, the ability of various kinases to phosphorylate the CT at the same time, and the inability to precisely control which residues are phosphorylated. Strategies employed to overcome these difficulties include the use of phospho-specific CT antibodies (7) and short CT phosphopeptides (8, 9). Also well appreciated is the use of Asp (or Glu) substitutions as a mimetic for phosphorylation (10, 11).The negative charge of the phosphate could affect the permeability of ions through the pore, alter the structure of the transmembrane ␣-helices to influence pore size, or modify the binding affinities of molecular partners involved in Cx43 regulation. Notably, if phosphorylation modifies protein interactions to affect the kinetics of channel assembly/disassembly or degradation, cell-to-cell communication will also be altered. Evidence has emerged that a phosphate can directly block the Cx43CT interaction with ZO-1 (9) and tubulin (8) or enhance the interaction with Nedd4 (12, 13). This information is critical because a particular cellular condition can be correlated with a specific Cx43 phosphorylation status to understand which proteins will and will not interact to affect regulation of Cx43. Numerous serine protein kinases have been identified as...

Regulation of Connexin32 by ephrin receptors and T-cell protein-tyrosine phosphatase

“…Of note, Cx32 residue Ser 233 is also associated with increased GJIC when phosphorylated (12). This finding is in contrast to tyrosine phosphorylation of the Cx43CT, as phosphorylation of Tyr 247 , Tyr 265 , and Tyr 313 is associated with decreased GJIC (10,(32)(33)(34)(35)(36). Taken together, this information suggests that phosphorylation (even by the same kinase) regulates different connexin isoforms via different mechanisms.…”

Altering integrin engagement regulates membrane localization of Kir2.1 channels

“…The surface biotinylation assay was performed following previously described methods (Li et al, 2016). Specifically, cells were plated in 10-cm tissue culture-treated dishes at 800,000 cells/dish and FAK inhibitor was added 2 h after plating.…”

Protein–Protein Interactions with Connexin 43: Regulation and Function

“…In addition to Src, another tyrosine kinase identified to directly interact with and phosphorylate the Cx43CT was the Janus kinase family member non-receptor tyrosine-protein kinase 2 (Tyk2; [ 125 ]). Interestingly, Tyk2 can functionally substitute for Src as work from our laboratory identified that it phosphorylates Cx43CT residues Y247 and Y265 and results in concomitant loss of coupling and disassembly of gap junction plaques [ 125 ].…”

“…In addition to Src, another tyrosine kinase identified to directly interact with and phosphorylate the Cx43CT was the Janus kinase family member non-receptor tyrosine-protein kinase 2 (Tyk2; [ 125 ]). Interestingly, Tyk2 can functionally substitute for Src as work from our laboratory identified that it phosphorylates Cx43CT residues Y247 and Y265 and results in concomitant loss of coupling and disassembly of gap junction plaques [ 125 ]. While phosphorylation of these sites by either Tyk2 or Src would result in disruption of the direct binding of β-tubulin and Drebrin, one difference is that Tyk2 unlikely disrupts the Cx43/ZO-1 interaction as Tyk2 does not contain a SH3 domain (for review see [ 126 ]).…”