Retinoic acid mediates degradation of IRS-1 by the ubiquitin-proteasome pathway, via a PKC-dependant mechanism.
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-delta 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-delta phosphorylates IRS-1 in vitro, (2) PKC-delta and IRS-1 interact in RA-treated cells, and (3) mutation of three PKC-delta 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.
del Rincón
Sonia V
SV
Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital and McGill University, Departments of Oncology and Medicine, Montreal, Quebec, Canada.
Guo
Qi
Q
Morelli
Catia
C
Shiu
Hoi-Ying
HY
Surmacz
Eva
E
Miller
Wilson H
WH
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D017931:DNA Primers; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014212:Tretinoin / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism*
D017931:DNA Primers; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011480:Protease Inhibitors; D025801:Ubiquitin; D014212:Tretinoin; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex