Phosphorylated Carboxy Terminal Serine Residues Stabilize the Mouse Gap Junction Protein Connexin45 Against Degradation

Abstract

Phosphoamino acid analysis of mouse connexin45 (Cx45) expressed in human HeLa cells revealed that phosphorylation occurred mainly at serine residues, but also on tyrosine and threonine residues. To characterize the role of Cx45 phosphorylation, different serine residues of the serine-rich carboxy terminal region were deleted or exchanged for other amino acids residues. Human HeLa cells deficient in gap junctional intercellular communication were stably transfected with appropriate constructs and analyzed for expression, localization, phosphorylation, formation of functional gap junction channels and degradation of mutant Cx45. fter exchange or deletion of nine carboxy terminal serine residues, phosphorylation was decreased by 90%, indicating that these serine residues represented main phosphorylation sites of mouse Cx45. The various serine residues of this region contributed differently to the phosphorylation of Cx45 suggesting a cooperative mechanism for phosphorylation. Substitution of different serine residues for other amino acids did not interfere with correct intracellular trafficking and assembly of functional gap junction channels, as shown by localization of mutant Cx45 at the plasma membrane and by dye transfer to neighboring cells. Truncated Cx45 was also weakly phosphorylated but was trapped in perinuclear locations. Dye transfer of these transfectants was similar as in nontransfected HeLa cells. The half-life of mouse Cx45 protein in HeLa cells was determined as 4.2 hr. Pulse-chase experiments with the different transfectants revealed an increased turnover of Cx45, when one or both of the serine residues at positions 381 and 382 or 384 and 385 were exchanged for other amino acids. The half-life of these mutants was diminished by 50% compared to wild type Cx45.

The effects of connexin phosphorylation on gap junctional communication

“…Cx45 has been shown to be phosphorylated in cells incubated with 32 Porthophosphate. Deletion of the last 26 amino acids of Cx45 (nine of which are serines) or substitution of the serines to glycine or alanine led to a 90% reduction in phosphorylation and a dramatic loss of communication in former case (Hertlein, Butterweck, Haubrich, Willecke, & Traub, 1998). Cx45 channels have also been shown to be modulated by phosphorylation .…”

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

“…HeLa cells were metabolically labeled with [ 35 S]methionine for 1 h, as described by Hertlein et al (1998). Medium was replaced by nonradioactive medium, supplemented with additional 15 mg/l methionine (final concentration 45 mg/l).…”

Casein Kinase II Phosphorylates Lens Connexin 45.6 and Is Involved in Its Degradation

“…In contrast, for mouse Cx45 it has been shown that decreased phosphorylation coincided with degradation of this protein (2). The substitution of one or more serine residues for other amino acids increases degradation of the transfected Cx45 in HeLa cells (5). Since in vivo phosphorylation sites for any of the above mentioned connexins have not been mapped, the direct relationship between in vivo phosphorylation and connexin degradation is not clear.…”

“…The COOH terminus, the most variable region among connexins, contains several putative kinase consensus sequences. The phosphorylation of connexins has been indicated in the intracellular trafficking of connexins (2), channel assembly (3), gating of gap junctional communication (4), stabilization of connexins from degradation (5,6), etc. In isolated myocardial gap junctions, most Cx43 phosphorylation sites are in the region 271-382 (7).…”