Abstract
Abstract: :
Purpose: Previously studies have shown that diabetes caused a decrease in gap junction activity in the lens. Gap junction activity can be regulated by many environmental factors such as drugs, stressed-related factors, and growth factors. Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress and insulin-like growth factor (IGF)–I has been shown to regulate cell proliferation, differentiation, metabolism, and apoptosis. The goal of these studies is to determine how PKC γ activity is controlled through LEDGF or IGF-I in normal LECs, and subsequently how this control of PKC γ is regulated through 14-3-3 proteins. Methods: A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF or IGF-I. Endogenous sn-1,2-diacylglycerol (DAG) was measured by detecting 32P-labelled phosphatidic acid. Physical and functional interactions between PLC γ and PKC γ, and 14-3-3 protein and PKC γ were determined by co-immunoprecipitation and phosphorylation assays. Results: Previously we have shown that LEDGF- or IGF-I-induced activation of PKC γ results in the translocation of PKC γ from the cytosol to the membrane, phosphorylation of Connexin 43, and a decrease in gap junction acitivity. The results of DAG assay showed that 25 ng/ml IGF-I and 10 ng/ml LEDGF increased endogenous DAG level. In addition, 10 ng/ml LEDGF can increase interaction between PKC γ and PLC γ and also increase phosphorylation of PLC γ using pPLC γ antibodies. In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein-protein interaction between PKC γ and 14-3-3 protein. Specifically, 14-3-3 ε, γ, and ζ have significant increase in protein interaction with PKC γ in the presence of 10-20 ng/ml LEDGF compared to control; however, only 14-3-3 γ and ζ showed an increase in protein interaction with PKC γ and 14-3-3 ε showed a decrease in interaction with PKC γ in the presence of 25 ng/ml IGF-I. The interaction of 14-3-3 isoform and PKC γ is dose- and time-dependent. Conclusions: Although 14-3-3 protein has been implicated in the regulation of several enzymatic pathways, its effect on PKC activation in LECs has not been investigated previously. These results demonstrate that LEDGF or IGF activate PKC γ and promote the interaction between PKC γ and 14-3-3 protein. The specificity of 14-3-3 ε to interact with PKC γ in the presence of LEDGF and not IGF-I will be the focus of our future studies. (NIH EY13421)