Dissection of the Molecular Basis of pp60v-src Induced Gating of Connexin 43 Gap Junction Channels

Abstract

Suppression of gap-junctional communication by various protein kinases, growth factors, and oncogenes frequently correlates with enhanced mitogenesis. The oncogene v-src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potential target of v-src, although v-src action has also been associated with changes in serine phosphorylation. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH-terminal domain led to an almost complete loss of response of Cx43 to v-src, but this was restored by coexpression of the independent COOH-terminal polypeptide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K+ channel inactivation. Surprisingly, we found that v-src mediated gating of Cx43 did not require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphorylation sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v-src and other mitogens, such as EGF and lysophosphatidic acid (LPA).

Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43

“…Impaired channel closure due to deletion of the last 125 amino acids of the C terminus could be rescued by coexpression of the C-terminal fragment in Xenopus oocytes (Morley et al, 1996). This gating mechanism also has been observed in the regulation of Cx43 gap junctional channels by insulin/insulin-like growth factor (Homma et al, 1998), platelet-derived growth factor (Moorby and Gherardi, 1999), v-src (Zhou et al, 1999), and transjunctional voltage (Moreno et al, 2002). Apart from this intramolecular protein-protein interaction, the C terminus of Cx43 can directly bind to other proteins.…”

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

“…The plasmid encoding the full‐length rat Cx43 (abbreviated as Wt) was provided by R. Civitelli (Washington University, Saint Louis, MO) (Lecanda et al ., 1998). The mutant Cx43 truncated at amino acid 245 (abbreviated as Cx43 Δ245 ) (Zhou et al ., 1999) and the Cx43 carboxy‐terminal tail (abbreviated as Cx43 C‐tail ) (Zhou et al ., 1999) were provided by B. Nicholson (University of Texas, Santo Antonio, TX). Cx43 lacking seven residues from the internal loop at positions 130–136 (abbreviated as Cx43 Δ130 ) was provided by V.A.…”

Is there a role for remodeled connexins in AF? No simple answers

“…Under pathological conditions Cx43 may be dephosphorylated or the phosphorylation may change from Serine/Threonine to Tyrosine phosphorylation, rather than a complete dephosphorylation of the protein [71]. For example, under conditions in which the non-receptor tyrosine kinase cSrc is activated, Cx43 loses Serine/ Threonine phosphoryations but gains a Tyrosine phosphorylation [72]. Depending on the phospho-residues in question, both loss of Serine Threonine phosphorylation as well as a gain of Tyrosine phosphorylation are associated with loss of cell-cell coupling [71,72].…”

“…For example, under conditions in which the non-receptor tyrosine kinase cSrc is activated, Cx43 loses Serine/ Threonine phosphoryations but gains a Tyrosine phosphorylation [72]. Depending on the phospho-residues in question, both loss of Serine Threonine phosphorylation as well as a gain of Tyrosine phosphorylation are associated with loss of cell-cell coupling [71,72]. It has been shown that phosphatases are activated in atrial fibrillation [73,74] but direct studies on the relationship between phosphatases and connexins in the fibrillating atrium have yet to be published.…”