Abstract
Gap junctions contain intercellular channels that enable intercellular communication of small molecules while also serving as a signaling scaffold. Connexins, the proteins that form gap junctions in vertebrates, are highly regulated and typically have short (<2 h) half-lives. Connexin43 (Cx43), the predominate connexin in the myocardium and epithelial tissues, is phosphorylated on more than a dozen serine residues and interacts with a variety of protein kinases. These interactions regulate Cx43 and gap junction formation and stability. Casein kinase 1 (CK1)-mediated phosphorylation of Cx43 promotes gap junction assembly. Using murine knock-in technology and quantitative PCR, immunoblotting, and immunoprecipitation assays, we show here that mutation of the CK1 phosphorylation sites in Cx43 reduces the levels of total Cx43 in the myocardium and increases Cx43 phosphorylation on sites phosphorylated by extracellular signal-regulated kinase (ERK). In aged myocardium, we found that, compared with WT Cx43, mutant Cx43 expression increases ERK activation, phosphorylation of Akt substrates, and protection from ischemia-induced injury. Our findings also uncovered that Cx43 interacts with the hypoxia-inducible protein N-Myc downstream-regulated gene 1 protein (NDRG1) and that Cx43 phosphorylation status controls this interaction and dramatically affects NDRG1 stability. We propose that, in addition to altering gap junction stability, Cx43 phosphorylation directly and dynamically regulates cellular signaling through ERK and Akt in response to ischemic injury. We conclude that gap junction-dependent NDRG1 regulation might explain some cellular responses to hypoxia.
Highlights
Gap junctions contain intercellular channels that enable intercellular communication of small molecules while serving as a signaling scaffold
We compared the extent of myocardial infarction in response to ischemia reperfusion injury (IRI) in hearts from young (3– 6 months old) and old (Ͼ12 months old; average, 16 months) mice expressing Cx43 with mutations at CK1 phosphorylation sites (Cx43CK1) compared with syngeneic with age (WT) mice
We found that the protocol caused partial ischemic damage but no difference in the area of living tissue between young WT and Cx43CK1 animals (Fig. 1B, 45.9% versus 44.4% tetrazolium chloride (TTC)-positive)
Summary
Critical signaling and scaffolding roles that can be fully or partially independent of channel function. ERK, Akt, and PKC are well known to modulate cell survival, in the myocardium, where they can regulate propagation and protect from ischemia reperfusion injury (IRI) (19 –21). Gap junction remodeling and phosphorylation of Cx43 by PKC and Akt occur in response to short-term ischemia in the myocardium [17, 22], and phosphorylation on Ser-262, a potential ERK site [12], has been suggested to promote cell survival and protection of the myocardium from IRI [23, 24]. Previous studies have shown that mutation of the Cx43 sites phosphorylated by CK1 to alanine in a knock-in mouse model (hereafter called Cx43CK1) results in animals that develop normally but have diminished expression of Cx43 in many tissues, including the heart (13, 26 –28). These findings indicate that Cx43-mediated effects on cell behavior are not merely downstream effects of kinase activation but, rather, may exert control over the kinases themselves
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