Polyubiquitination and proteasomal degradation of the p185(c-erbB-2) receptor protein-tyrosine kinase induced by geldanamycin

Treatment of SKBr3 human breast carcinoma cells with the benzoquinoid ansamycin, geldanamycin, rapidly depletes p185(c-erbB-2) protein-tyrosine kinase. Loss of p185(c-erbB-2) is initiated by disruption of a heteromeric complex between p185(c-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 p185(c-erb-2) becomes polyubiquitinated. Treatment of cells with the specific proteasome proteolytic inhibitor, lactacystin, blocked geldanamycin-induced degradation of p185(c-erbB-2) and enhanced the accumulation of polyubiquitinated p185(c-erbB-2). Following geldanamycin and lactacystin treatment, a higher molecular weight form of p185(c-erbB-2), which likely represents ubiquitin-p185(c-erbB-2) conjugates, was detected by anti-p185(c-erbB-2) immunoblotting. Nascent p185(c-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 p185(c-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.