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Retinoic acid mediates degradation of IRS-1 by the ubiquitin–proteasome pathway, via a PKC-dependant mechanism

Oncogene volume 23, pages 9269–9279 (2004)Cite this article

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-δ 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-δ phosphorylates IRS-1 in vitro, (2) PKC-δ and IRS-1 interact in RA-treated cells, and (3) mutation of three PKC-δ 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.

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Acknowledgements

We are grateful to Dr Chris Sell for providing all FLAG-tagged IRS-1 constructs used in this manuscript. We thank Dr Dirk Bohmann for providing the HA-tagged ubiquitin construct. We extend our kindest thanks to Dr Richard A Roth for providing the GST-IRS-1288−678WT and GST-IRS-1288−678MUT constructs. We also thank Dr C Ronald Khan for his generous gift of human IRS-1 cDNA. This work was supported by a predoctoral fellowship award from the US Army Medical Research and Materiel Command Breast Cancer Research Program (Award number DAMD1701-1-0320 to SV del Rincón) and a grant from the Canadian Breast Cancer Research Initiative. Wilson H Miller Jr is an Investigator of the Canadian Institutes of Health Research.

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Authors and Affiliations

  1. Departments of Oncology and Medicine, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital and McGill University, Montreal, Quebec, Canada

    Sonia V del Rincón, Qi Guo, Hoi-Ying Shiu & Wilson H Miller Jr

  2. Postgraduate School in Clinical Pathology, University of Calabria, Cosenza, Italy

    Catia Morelli

  3. Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, 19107, PA, USA

    Eva Surmacz

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  1. Sonia V del Rincón
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Correspondence to Wilson H Miller Jr.

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Rincón, S., Guo, Q., Morelli, C. et al. Retinoic acid mediates degradation of IRS-1 by the ubiquitin–proteasome pathway, via a PKC-dependant mechanism. Oncogene 23, 9269–9279 (2004). https://doi.org/10.1038/sj.onc.1208104

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