Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning, ultrastructural localization, and post-translational phosphorylation

Abstract

Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with 32P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.

Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly

“…Is Phosphorylated by CK1␦ in Vitro and in Cell Culture-Several studies have shown that Cx43 is phosphorylated on multiple different serine residues throughout its life cycle in homeostatic cells (12,33,34). Many kinases including CK1 are thought to remain constitutively active in cells and could phosphorylate Cx43 in the absence of exogenous kinase stimulation.…”

Cx31 is assembled and trafficked to cell surface by ER-Golgi pathway and degraded by proteasomal or lysosomal pathways

“…Unlike many other membrane proteins, connexins are relatively dynamic, for pulse-chase assays had demonstrated that most connexins have half-lives of 1-3 h in cultured cells [12][13][14][15]. Turnover, degradation, and remodeling of gap junctions may contribute substantially to the regulation of intercellular communication [16][17][18].…”

Expression of connexin48.5, connexin44.1, and connexin43 during zebrafish (Danio rerio) lens development

“…Cx46 and Cx50 are mainly expressed in the lens fiber cells (Paul et al, 1991;White et al, 1992), whereas Cx43 is restricted to the anterior lens epithelium (Yancey et al, 1992). The chicken orthologues of these connexins have been identified as Cx56, Cx45.6, and Cx43 (Musil et al, 1990;Rup et al, 1993;Jiang et al, 1994). Both Cx46 and Cx50 knockout mice developed cataracts (Gong et al, 1997;White et al, 1998), indicating the importance of gap junctions in the maintenance of lens fiber homeostasis in mammals.…”