Exchange of serine residues 263 and 266 reduces the function of mouse gap junction protein connexin31 and exhibits a dominant-negative effect on the wild-type protein in HeLa cells.
To characterize the role of Cx31 phosphorylation, serine residues 263 and 266 (Cx31Delta263,266) or 266 (Cx31Delta266) alone were exchanged for amino acids that cannot be phosphorylated. HeLa cells, which were stably transfected with wild type and the two different mutant Cx31-cDNA constructs, were analyzed for expression, phosphorylation, localization, formation of functional gap junction channels, and degradation of mutant Cx31 protein. Both mutant proteins showed similar reduced phosphorylation levels compared to Cx31 wild type, indicating a pivotal role of serine residue 266 for Cx31 phosphorylation. None of these mutations did interfere with correct intracellular trafficking of gap junction proteins. Pulse chase experiments with the different transfectants revealed an increased turnover of both mutated Cx31 proteins. They showed decreased intercellular communication as shown by dye transfer to neighboring cells and measurement of total conductance (mutant Cx31Delta263,266). Mutated Cx31 protein (Cx31Delta263,266) diminished the function of the Cx31 wild-type protein dependent on the amount of the mutated protein, indicating a dominant-negative effect of the mutated protein in HeLa cells.
Diestel
Simone
S
Department of Biochemistry, Institute of Animal Anatomy and Physiology, University of Bonn, Bonn 53115, Germany.
Eckert
Reiner
R
Hülser
Dieter
D
Traub
Otto
O
D000595:Amino Acid Sequence / Q000235:genetics*; D000818:Animals; D002450:Cell Communication / Q000235:genetics; D017630:Connexins / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000235:genetics; D051379:Mice; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation; D021381:Protein Transport / Q000235:genetics; D012694:Serine / Q000235:genetics*; D014162:Transfection
D017630:Connexins; D018076:DNA, Complementary; C068044:GJB3 protein, human; D012694:Serine