PHOSPHORYLATED CARBOXY-TERMINAL SERINE RESIDUES STABILIZE THE MOUSE GAP JUNCTION PROTEIN CONNEXIN45 AGAINST DEGRADATION

Phosphoamino acid analysis of mouse connexin45 (Cx45) expressed in hum an 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 se rine-rich carboxy terminal region were deleted or exchanged for other amino acids residues. Human HeLa cells deficient in gap junctional int ercellular communication were stably transfected with appropriate cons tructs and analyzed for expression, localization, phosphorylation, for mation of functional gap junction channels and degradation of mutant C x45. After exchange or deletion of nine carboxy terminal serine residu es, phosphorylation was decreased by 90%, indicating that these serine residues represented main phosphorylation sites of mouse Cx45. The va rious ser ine residues of this region contributed differently to the p hosphorylation of Cx45 suggesting a cooperative mechanism for phosphor ylation. Substitution of different serine residues for other amino aci ds did not interfere with correct intracellular trafficking and assemb ly of functional gap junction channels, as shown by localization of mu tant Cx45 at the plasma membrane and by dye transfer to neighboring ce lls. 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 t he different transfectants revealed an increased turnover of Cx45, whe n one or both of the serine residues at positions 381 and 382 or 384 a nd 385 were exchanged for other amino acids. The half-life of these mu tants was diminished by 50% compared to wild type Cx45.