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V.J. Kuonen, N. Chaturvedi, C.M. Colitz; Phosphorylation of Telomerase in Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4510.
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Introduction: Telomerase is a ribonucleoprotein complex responsible for maintenance of telomere length and prevention of chromosomal degradation and recombination and repair of DNA strand breaks. We previously found telomerase activity in normal and cataractous lens epithelial cells. In other cell types with telomerase activity, post-translational phosphorylation by protein kinase C plays a major role in telomerase regulation. Our goal was to determine which protein kinase C isoform was responsible for phosphorylation of telomerase in lens epithelial cells. Methods: Primary canine lens epithelial cell cultures grown in laminin-coated flasks in complete media (DMEM, 10% fetal bovine serum, antibiotic/antimycotic) were passed once into 12-well laminin coated plates until they reached 60% confluence. Cells were treated with two different protein kinase C inhibitors (Go6976 at 60, 100, and 200nM for 24 or 48 hours and Go6983 at 40, 60 or 120 uM for 15 or 24 hours). There were two untreated control wells per experiment. Cells were then harvested, protein extracted with Chaps buffer and quantified by Bradford assay and then 2ug protein was used in the telomeric repeat amplification protocol-ELISA assay (Roche) to determine telomerase activity. Data is in OD units measured at 450 nm with reference of 690 nm. Results: Go6976 did not significantly inhibit telomerase activity at any concentration or time period. Go6983 inhibited telomerase activity at 60 uM with a 15 hour incubation (TA=0.068, control 0.229; p value=0.0014). Conclusions: Protein kinase C is a cyclic-nucleotide independent enzyme that phosphorylates serine and threonine residues. To date, ten PKC isoforms have been identified. Various PKC isoforms phosphorylate telomerase in different telomerase positive cell types. Different chemical PKC inhibitors have been used to prove this. Go6976 inhibits isoforms alpha and beta. Go6983 inhibits PKC isoforms alpha, beta, gamma, sigma at lower concentrations, and zeta at higher concentrations. Thus far only Go6983 at 60uM, but not 40 or 120uM has inhibited telomerase activity, implying that PKC zeta is responsible for phosphorylating telomerase in LEC.
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