Abstract
Purpose: :
The purpose of this study is to determine the PKCγ phosphorylation site on Cx50 in lens epithelial cells and subsequent functional results of phosphorylation.
Methods: :
Mutation (S430A) was introduced into the wild type Cx50:EGFP by site–directed mutagenesis. Wild–type and mutated (S430A) Cx50 were transfected into 80% confluent N/N lens epithelial cells, and stably transfected cells were selected in DMEM media. PKCγ was activated by phorbol–12 myristate 13 acetate (TPA, 200nM). Expression and localization of wild type and mutated Cx50–EGFP fusion proteins before and after TPA treatment were measured by confocal microscopy. Cell surface Cx50 gap junction plaques were immuno–labeled and counted by confocal microscopy. Co–localization of Cx50 and PKCγ was determined by co–immunoprecipitation. Phosphorylation of Cx50–S430 was determined by western blotting with anti–phosphoserine antibodies and functional effects were measured by gap junction plaque assembly–disassembly.
Results: :
Cx50S430A localization was similar to the wild type and had punctate membrane localization at cell/cell contacts. PKCγ activation by TPA resulted in disassembly of wild type Cx50 plaques, but this had no effect on the Cx50S430A mutant. PKCγ was co–localized with both wild type Cx50 and the mutant S430ACx50 demonstrating similar interactions in both Cx50s.
Conclusions: :
Cx50 is phosphorylated at S430 by PKCγ and this results in Cx50 gap junction disassembly.
Keywords: gap junctions/coupling • microscopy: confocal/tunneling • phosphorylation