Abstract
Purpose: :
Investigate the effects of PKC phosphorylation on the water permeability of AQP0.
Methods: :
Xenopus oocyte water permeability assay, BtAQP0, site directed mutagenesis, PKC activation, PKC inhibition, Fluorescent Resonance Energy Transfer (FRET), immunohistochemistry.
Results: :
AQP0, the major intrinsic protein of lens fiber cells, can act as a water channel, junctional protein, or anchor protein for the cytoskeleton and promotes the formation of micro-domains in the plasma membrane. During fiber cell differentiation AQP0 undergoes spatially and temporally regulated phosphorylations that could have unique physiological roles in lens fiber cells. The pattern of C-terminal phosphorylation of AQP0 creates four distinct water permeability (Pf) phenotypes: 1. Low-Pf with 0 mM Ca2+ sensitivity; 2. Low-Pf with 5 mM Ca2+ sensitivity; 3. Low-Pf no Ca2+ sensitivity; 4. High-Pf with no Ca2+ sensitivity (Kalman, K. et al. .JBC., 2008). In the inner cortex, Ser229 and Ser231 phosphorylation increases (Ball, L.E. et al. Biochem. 2004), and these modified AQP0s become calmodulin-independent high-Pf-phenotype channels. Using site-directed mutagenesis, PKC activators and inhibitors, we investigated the effects of PKC phosphorylation(s) on "group-4" Ser229Asp, Ser229Asn, Ser231Asp and Ser231Asn mutants. Our data show that DAG-activated PKC inhibits the Pf of Ser229Asp mutant and probably to closes the channel pore. This result reveals a fifth phenotype of regulation of AQP0 by phosphorylation.
Conclusions: :
PKC phosphorylation is an essential component of the regulation of AQP0 water permeability. Our observation suggests that there is a particular combination of C-terminus serine-phosphorylation which completely eliminates AQP0 water permeability. This suggests a greater range of Pf regulation then before observed.
Keywords: cataract • aging • phosphorylation