Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

Abstract

The carboxyl-terminal (CT) domain of connexin43 (Cx43) has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione Stransferase (GST)-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion proteincoated beads. The bound proteins were washed and then incubated with 32 P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43. Correspondence

pH-Dependent Intramolecular Binding and Structure Involving Cx43 Cytoplasmic Domains

“…This vector contains the coding region of glutathione S-transferase (GST) fused to the cleavage site for human rhinovirus 3C protease (see below). The template was produced as previously described (15). To clone the Cx43CT domain, the following primers were used: forward, GAA GGA TCC ATG AGC GAT CCT TAC CAC GCC; and reverse, GCT TGA ATT CCA AGC CGG TTT AAA TCT CC.…”

“…Production of 15 N-Labeled Cx43CT-Bacteria were grown as indicated above, but in a minimal medium containing 100 mM KH 2 …”

“…Gradient-enhanced two-dimensional 1 H 15 N HSQC experiments (26) were used to observe all backbone amide resonances in 15 N-labeled Cx43CT. Data were acquired with 1024 complex points in the direct dimension and 64 complex points in the indirect dimension.…”

“…Sweep widths were 8389 Hz in the proton dimension and 2432 Hz in the nitrogen dimension. Amide proton-amide proton NOEs were observed in two-dimensional 15 N NOESY-HSQC spectra (26) collected with 512 and 2048 complex points, for t 1 (H) and t 2 (HN) time domains, and a mixing time of 125 ms.…”

Biochemical Requirements for Inhibition of Connexin26-containing Channels by Natural and Synthetic Taurine Analogs

“…For these connexins, pH dependence may require soluble factors, such as aminosulfonates or pH-dependent kinase activity (67) or direct protonation of other domains, as for Cx46 (68). In addition, there is evidence that pH regulation may differ among species orthologs and in different expression systems (69,70).…”