Research article

Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells

Anna L Stratford¹ , Christopher J Fry² , Curtis Desilets² , Alastair H Davies¹ , Yong Y Cho³ , Yvonne Li¹ , Zigang Dong³ , Isabelle M Berquin⁴ , Philippe P Roux⁵ and Sandra E Dunn¹

¹  Laboratory for Oncogenomic Research, Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada

²  Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, USA

³  Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA

⁴  Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA

⁵  Department of Pathology and Cell Biology, Faculty of Medicine, Institute for Research in Immunology and Cancer, P.O. Box 6128, Station Centre-Ville, Université de Montréal, Montreal, QC H3C 3J7, Canada

author email corresponding author email

Breast Cancer Research 2008, 10:R99doi:10.1186/bcr2202

Published:	27 November 2008

Abstract

Introduction

Basal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1.

Methods

Inhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation.

Results

Using an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2^-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not.

Conclusions

We therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention.