May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Protein Kinase C Regulates Gap Junction Activity after Release from 14–3–3
Author Affiliations & Notes
  • T.A. Nguyen
    Biochemistry, Kansas State University, Manhattan, KS
  • D.J. Takemoto
    Biochemistry, Kansas State University, Manhattan, KS
  • Footnotes
    Commercial Relationships  T.A. Nguyen, None; D.J. Takemoto, None.
  • Footnotes
    Support  NIHEY13421
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3984. doi:
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      T.A. Nguyen, D.J. Takemoto; Protein Kinase C Regulates Gap Junction Activity after Release from 14–3–3 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3984.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: The decrease in PKC γ levels during galactosemia and in human diabetic lens would result in an increase in gap junction activity. Previously evidence has shown that growth factors such as IGF or LEDGF activate the translocation of PKC γ to the membrane, interaction of PKCγ and Cx43, and phosphorylation of Cx43, and subsequently decrease of gap junction activity. PKC γ–mediated phosphorylation of Cx43 and subsequent decrease of gap junction activity could be through the release of PKC γ from 14–3–3, a cytosolic docking protein, followed by the activation of PKC γ and subsequent PKC γ–catalyzed phosphorylation of Cx43. In this study, we will investigate the interaction of PKC γ and 14–3–3 and determine how the release of endogenous PKCγ subsequently decreases gap junction activity in lens epithelial cells. Methods: A rabbit lens epithelial, N/N1003A, cell line was grown in the absence and presence of growth factors (IGF or LEDGF) or synthetic peptides. Endogenous PKC γ total activity and gap junction activity were determined by a PKC enzyme assay and scrape load / dye transfer assay, respectively. Physical interactions between PKCγ and 14–3–3 were determined by co–immunoprecipitation assays. Results: We have identified two sites of PKC γ that are responsible for the interaction with 14–3–3 during the inactive state of PKC γ. Two sites, C1B1 and C1B5, are located within the C1B– domain (residues 101–150) of PKC γ. The presence of C1B1 (residues 101–112) and/or C1B5 (residues 141–150) can directly compete for the binding of 14–3–3 to endogenous PKC γ resulting in the release of endogenous PKC γ, activation of enzyme activity, and subsequent translocation to the membrane. 100 µM of C1B1, C1B5, or both peptides for 2 hrs is sufficient to compete for the binding of 14–3–3 and cause the release of endogenous PKC γ from 14–3–3; however, these peptides did not altered the interaction of raf–1 and 14–3–3. Additionally, the results with 100 µM of C1B1, C1B5, or both peptides for 2 hrs in cells, using scrape loading/ dye transfer assay, show a significant decrease in gap junction activity. Conclusions: These results suggest that the activity and localization of PKC γ is regulated by the binding to 14–3–3. The PKC γ binding sites are within the C1B–domain of PKC γ. Synthetic peptides from the C1B–domain of PKCγ can be used to inhibit gap junction activity.

Keywords: gap junctions/coupling 
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