Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells.
BACKGROUND
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, PKC alpha. 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.
Stratford
Anna L
AL
Laboratory for Oncogenomic Research, Department of Pediatrics, Child and Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada.
Fry
Christopher J
CJ
Desilets
Curtis
C
Davies
Alastair H
AH
Cho
Yong Y
YY
Li
Yvonne
Y
Dong
Zigang
Z
Berquin
Isabelle M
IM
Roux
Philippe P
PP
Dunn
Sandra E
SE
D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D009005:Monosaccharides / Q000494:pharmacology; D018295:Neoplasms, Basal Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D051840:Y-Box-Binding Protein 1 / Q000235:genetics / Q000378:metabolism*
D001578:Benzopyrans; D009005:Monosaccharides; D034741:RNA, Small Interfering; C497449:SL0101; D051840:Y-Box-Binding Protein 1; D012694:Serine; D008156:Luciferases; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D051571:Protein Kinase C-alpha