1. ¹Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital and McGill University, Departments of Oncology and Medicine, Montreal, Quebec, Canada
2. ²Postgraduate School in Clinical Pathology, University of Calabria, Cosenza, Italy
3. ³Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA

Correspondence: WH Miller Jr, Lady Davis Institute, 3755 Cote Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2. E-mail: wmiller@ldi.jgh.mcgill.ca

Received 15 April 2004; Revised 7 July 2004; Accepted 17 August 2004; Published online 1 November 2004.

Abstract

Insulin receptor substrate-1 (IRS-1) mediates signaling from the insulin-like growth factor type-I receptor. We found that all-trans retinoic acid (RA) decreases IRS-1 protein levels in MCF-7, T47-D, and ZR75.1 breast cancer cells, which are growth arrested by RA, but not in the RA-resistant MDA-MB-231 and MDA-MB-468 cells. Based on prior reports of ubiquitin-mediated degradation of IRS-1, we investigated the ubiquitination of IRS-1 in RA-treated breast cancer cells. Two proteasome inhibitors, MG-132 and lactacystin, blocked the RA-mediated degradation of IRS-1, and RA increased ubiquitination of IRS-1 in the RA-sensitive breast cancer cells. In addition, we found that RA increases serine phosphorylation of IRS-1. To elucidate the signaling pathway responsible for this phosphorylation event, pharmacologic inhibitors were used. Two PKC inhibitors, but not a MAPK inhibitor, blocked the RA-induced degradation and serine phosphorylation of IRS-1. We demonstrate that RA activates PKC- in the sensitive, but not in the resistant cells, with a time course that is consistent with the RA-induced decrease of IRS-1. We also show that: (1) RA-activated PKC- phosphorylates IRS-1 in vitro, (2) PKC- and IRS-1 interact in RA-treated cells, and (3) mutation of three PKC- serine sites in IRS-1 to alanines results in no RA-induced in vitro phosphorylation of IRS-1. Together, these results indicate that RA regulates IRS-1 levels by the ubiquitin–proteasome pathway, involving a PKC-sensitive mechanism.