Differential phosphorylation of connexin46 and connexin50 byH2O2 activation of protein kinase Cγ

[摘要] Purpose: Fiber cell gap junction proteins connexin 46 (Cx46) andconnexin 50 (Cx50) play distinct roles in the avascular lens. Thepurpose of this study was to determine how protein kinase Cγ(PKCγ) differentially regulates phosphorylation of Cx46 and Cx50 inoxidatively stressed lenses.Methods: Sprague Dawley rats (six week old) were used in theexperiments. PKCγ enzyme activity was analyzed by use of thePepTag assay kit. Phosphorylation of caveolin-1, Cx46, and Cx50 wasdetermined by immunoblotting. Lipid rafts were isolated by continuoussucrose gradient centrifugation. Lipid raft-localization of PKCγ,Cx46, or Cx50 was demonstrated by immunoblotting. Association ofcaveolin-1 with PKCγ, Cx46, or Cx50 was revealed byco-immunoprecipitation.Results: H2O2 (100 μM) stimulated PKCγactivation in rat whole lens. Activated PKCγ was recruited intocaveolin-1 (Cav-1) containing lipid rafts and this activation enhancedthe coimmunoprecipitation of Cav-1, Cx46, and Cx50 with PKCγ. BothCx50 and Cx46 were associated with Cav-1 in lipid rafts.H2O2 significantly induced threonine (Thr) phosphorylationof Cx46 and Cx50, and serine (Ser) phosphorylation of Cx50. However,There was only a small stimulation of Cx46 phosphorylation at Ser byH2O2, as Cx46 was already phosphorylated.Conclusions: Activation of PKCγ by H2O2 stimulateddifferential Ser phosphorylation of Cx50 versus Cx46, within lipidrafts. This suggests that Cx50 and Cx46 may have different functions inlens.

[学科分类] 生物化学/生物物理