Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin.
Treatment of SKBr3 human breast carcinoma cells with the benzoquinoid ansamycin, geldanamycin, rapidly depletes p185c-erbB-2 protein-tyrosine kinase. Loss of p185c-erbB-2 is initiated by disruption of a heteromeric complex between p185c-erbB-2 and the 94-kDa glucose-regulated protein, GRP94, to which geldanamycin binds avidly. Here we report that within minutes of exposure to geldanamycin, mature p185c-erbB-2 becomes polyubiquitinated. Treatment of cells with the specific proteasome proteolytic inhibitor, lactacystin, blocked geldanamycin-induced degradation of p185c-erbB-2 and enhanced the accumulation of polyubiquitinated p185c-erbB-2. Following geldanamycin and lactacystin treatment, a higher molecular weight form of p185c-erbB-2, which likely represents ubiquitin-p185c-erbB-2 conjugates, was detected by anti-p185c-erbB-2 immunoblotting. Nascent p185c-erbB-2 synthesized in the presence of geldanamycin is incompletely glycosylated and remains sequestered in the endoplasmic reticulum. While this immature form of the protein is not ubiquitinated in the presence of geldanamycin, its marked, drug-induced instability is nonetheless antagonized by lactacystin. Thus, the rapid depletion of mature p185c-erbB-2 caused by geldanamycin and the marked, drug-stimulated decrease in half-life of the newly synthesized protein are both mediated by the proteasome, although only the former phenomenon involves polyubiquitination.
Mimnaugh
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EG
Clinical Pharmacology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Chavany
C
C
Neckers
L
L
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D016227:Benzoquinones; D001704:Biopolymers / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D047029:Lactams, Macrocyclic; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011809:Quinones / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D017828:Rifabutin / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism*
D000111:Acetylcysteine; D016227:Benzoquinones; D001704:Biopolymers; D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D009097:Multienzyme Complexes; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011809:Quinones; D018719:Erb-b2 Receptor Tyrosine Kinases; D017828:Rifabutin; D014452:Ubiquitins; C067713:lactacystin; C001277:geldanamycin