Polyubiquitination and Proteasomal Degradation of the p185c-erbB-2 Receptor Protein-tyrosine Kinase Induced by Geldanamycin

doi:10.1074/jbc.271.37.22796

Polyubiquitination and Proteasomal Degradation of the p185^c-erbB-2 Receptor Protein-tyrosine Kinase Induced by Geldanamycin*

From the Clinical Pharmacology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

^‡ To whom correspondence should be addressed:
Clinical Pharmacology Branch, National Cancer Institute, National Institutes of Health, Bldg. 10, Room 12N226, Bethesda, MD 20892.
Tel.: 301-496-8050; Fax: 301-402-1608.

Abstract

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-erbB-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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

HA

herbimycin

GA

geldanamycin

GRP94

94-kDa glucose-regulated protein

HSP90

90-kDa heat shock protein

LC

lactacystin

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis.
↵²L. Guarino, personal communication.
- Received January 19, 1996.
- Revision received May 9, 1996.