Degradation of methoxysuccinyl-phe-leu-phe-7-amido-4-trifluoromethyl coumarin (FLF) in cultured myotubes and HepG2 cells is proteasome- and calpain/calcium-dependent

During recent years, it has become increasingly clear that the ubiquitin-pr oteasome proteolytic pathway regulates intracellular protein degradation in various physiological and pathophysiological conditions. Substrates specif ically degraded by the proteasome are important tools to assess the involve ment of the proteasome in cellular proteolysis. It was recently proposed th at the membrane permeable substrate methoxysuccinyl-phenylalanine-leucine-p henylalanine-7-amido-4- trifluoromethyl coumarin (FLF) is degraded specific ally by the proteasome. The role of other proteolytic pathways in the degra dation of FLF, however, is not fully understood. In the present study, we t ested the role of different proteolytic pathways in the degradation of FLF in cultured myotubes and HepG2 cells by treating the cells with inhibitors of lysosomal, calpain and proteasome activity. In addition, we tested the h ypothesis that insulin blocks proteasome-dependent degradation of FLF in my otubes and HepG2 cells. Results suggest that degradation of FLF in both myo tubes and HepG2 cells is regulated by proteasome and calpain activity but n ot by lysosomal activity. Insulin inhibited proteasome-dependent but not ca lpain-dependent degradation of FLF in both myotubes and HepG2 cells. The re sults are important because they suggest that FLF degradation does not spec ifically reflect proteasome activity. (C) 2000 Published by Elsevier Scienc e Ltd. All rights reserved.