Dingbo ARVO 2003

							CAVEOLIN REGULATION OF LENS EPITHELIAL CELL GAP JUNCTIONS
THROUGH PKC

D. Lin1, J. Zhou2, P.S. Zelenka2, D.L. Boyle3, and D.J. Takemoto1
1
  Department of Biochemistry, Kansas State University, Manhattan, KS 66506, 2 Laboratory of
Molecular and Development Biology, National Eye Institute, National Intitutes of Health,
Bethesda, MD 20892, 3 Division of Biology, Kansas State University, Manhattan, KS 66506

Purpose: Caveolins, principle structural proteins of the caveolae, mediate transmembrane
signaling through direct interaction with diverse signaling molecules. Cell-cell gap junctional
communications are regulated by environmental stimuli in the lens epithelial cells. Phorbol ester
and growth factors can regulate lens gap junctions by activation of PKC , which in turn cause
phosphorylation of connexin 43 and disassembly of gap junctions. This study demonstrates
interactions of caveolin-1 with PKC and connexin43, and its regulation in gap junctions in
response to growth factors.

Methods: N/N 1003A lens epithelial cells, primary bovine lens epithelial cells, and the stably
transfected N/N 1003A lens epithelial cells overexpressing PKC:GFP and point mutants as
fusion proteins were used for the experiments. Western blotting was employed to investigate the
expression of GFP fusion proteins and translocation of PKC. Cell surface gap junction Cx43
plaques, and colocalization of Cx43 and caveolin-1, were detected by confocal microscopy. Co-
immunoprecipitation was performed to determine the in vivo protein-protein interactions.
Redistribution of caveolin, PKC and Cx43 in response to growth factors was analyzed by
isolation of detergent-resistant membranes on sucrose gradients and by consequent Western
blotting.

Results: Caveolin1 and 2 but not caveolin 3, was found in N/N1003A and primary bovine lens
epithelial cells. TPA and IGF-I stimulated the interactions between caveolin 1 and PKC.
However Cx43 was always associated with caveolin 1. TPA and IGF-I induced redistribution of
caveolin1 and Cx43 from light density fractions to higher density fractions, indicating movement
out of caveolae “lipid rafts”. PKC::GFP fusion proteins overexpressed in N/N 1003A cells
translocated to plasma membranes in the presence of TPA or IGF-I. Overexpression of PKC
increased the interaction between PKC:GFP fusion protein and caveolin 1 or Cx43, and
decreased gap junctional Cx43 plaques in N/N 1003A LEC without exogenous growth factors.
However, overexpression of loss-of-function PKC mutants(activation loop or phosphorylation
site mutations) did not decrease gap junctions, even though PKC still interacted with caveolin 1
and Cx43.

Conclusion: Activation of PKC by TPA or IGF-I stimulated the interaction of PKC with
caveolin1 and Cx43, both of which were initially colocalized in detergent resistant lipid rafts.
This activation of PKC caused Cx43, caveolin 1 and PKC to redistribution within lipid rafts.
Use of loss-of-function PKC mutants suggests that PKC activity is required for gap junctions
movement within lipid rafts
   Supported by the grant of NIH EY13421.