Figure 4

Effect of PKC phosphorylation on telomerase holoprotein integrity and the enzyme activity. (A) After treatment with 10 μ M SC-10, PKC activity was determined by examining the amount of PKC-phosphosubstrates using pan-antibody immunoblot analysis. (B) Association study of hTERT-hsp90 and the influence of PKC phosphorylation. Lanes 1 and 2: Nuclear proteins from OEC-M1 cells with or without PKC-RNAi plasmid transfection were extracted and subjected to immunoprecipitation and immunoblot. Lanes 3 to 4: OEC-M1 cells were treated with 300 μ M novobiocin for 24 h to disrupt the hsp-hTERT association. Cells were harvested or continuously cultured with 10 μ M SC-10 for an additional 24 h (novobiocin+SC-10). Nuclear proteins were subjected to immunoprecipitation by hsp90 followed by immunoblot by hTERT or hsp90 (as control). (C) Alterations of telomerase activity after hTERT-hsp90 disruption and reassociation. Lanes 1 and 2: Cellular proteins from OEC-M1 cells with or without PKC-RNAi plasmid transfection were extracted for determination of telomerase activity by TRAP-EIA. Lanes 3 to 4: OEC-M1 cells were treated with 300 μ M novobiocin for 24 h to disrupt the hsp-hTERT association. Cells were harvested or continuously cultured with 10 μ M SC-10 for an additional 24 h. Cellular proteins were extracted for determination of telomerase activity.