leupeptins and rottlerin

Epidermal growth factor (EGF) receptor-overexpressing p53-deficient A431 cells response to toxic dose of EGF by G1 arrest and apoptosis was studied. We previously reported an increased expression of growth arrest and DNA-damage-inducible gene, Gadd45, in EGF-overexposed A431 cells. The mechanism for this induction was increased half-lives of mRNA and protein. In this study, using phorbol ester (a PKC activator) and specific inhibitors of PKC isoforms, we showed that protein kinase C-delta (PKCdelta) was involved in the increase of Gadd45 protein stability. We further demonstrated that Gadd45 is ubiquitinated and is regulated by proteolysis. While EGF induced ubiquitination of total cellular proteins, there was a decrease in Gadd45 ubiquitination, which could be inhibited by Rottlerin, a PKCdelta-specific inhibitor. These results suggest that an increase in Gadd45 stability may involve PKCdelta-dependent ubiquitin-proteasome pathway.

Topics: Acetophenones; Benzopyrans; Cell Division; Cycloheximide; DNA Damage; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; GADD45 Proteins; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Isoenzymes; Kinetics; Leupeptins; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Proteins; Pyridines; Recombinant Proteins; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Ubiquitins