Phosphorylated carboxy terminal serine residues stabilize the mouse gap junction protein connexin45 against degradation.
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.
Hertlein
B
B
Institut fuer Genetik, Abt. Molekulargenetik, Universitaet Bonn, Germany.
Butterweck
A
A
Haubrich
S
S
Willecke
K
K
Traub
O
O
D000818:Animals; D002264:Carboxylic Acids / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D017630:Connexins / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007424:Intracellular Fluid; D051379:Mice; D016296:Mutagenesis; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D012694:Serine / Q000378:metabolism*
D002264:Carboxylic Acids; D017630:Connexins; C082920:connexin 45; D010768:Phosphoserine; D012694:Serine