LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells.
Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress. The objectives of these studies are to determine how LEDGF controls PKC gamma activity in normal LECs: how this control of PKC gamma regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in LECs. A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF. PKC gamma protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng ml(-1). In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein-protein interaction between PKC gamma and Connexin 43. In the presence of LEDGF the activation of PKC gamma enhanced the phosphorylation of Connexin 43 by four-fold compared to the absence of LEDGF. The addition of LEDGF for 30 min resulted in a 65% decrease in gap junction Connexin 43 at the cell surface and a 70% decrease in gap junction activity. These results suggest that the activation of PKC gamma by LEDGF plays a major role in gap junction assembly/disassembly, which may enhance survival of LECs against osmolarity-stress induced by high sugar concentration.
Nguyen
Thu Annelise
TA
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Boyle
Daniel L
DL
Wagner
Lynn M
LM
Shinohara
Toshimichi
T
Takemoto
Dolores J
DJ
D000818:Animals; D018031:Connexin 43 / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000201:enzymology*; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D014178:Translocation, Genetic
D018031:Connexin 43; D036341:Intercellular Signaling Peptides and Proteins; C119380:lens epithelium-derived growth factor; D012694:Serine; C087857:protein kinase C gamma; D011493:Protein Kinase C