In vivo degradation of N-myc in neuroblastoma cells is mediated by the 26S proteasome.
N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.
Bonvini
P
P
Department of Cell and Cancer Biology, Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nguyen
P
P
Trepel
J
J
Neckers
L M
LM
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D009447:Neuroblastoma; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D014452:Ubiquitins; C067713:lactacystin; D010447:Peptide Hydrolases; D002154:Calpain; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000111:Acetylcysteine