Development of kinomic analyses to identify dysregulated signaling pathways in cells expressing cytoplasmic PrP

Virology Journal

Luis M Schang²  Xavier Roucou¹  Guillaume Tremblay¹  Catherine Grenier¹  Cathy Appanah³  Rory H Shott³ 

[1] Department of Biochemistry, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, QC J1H 5 N4, Canada;Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada;Department of Biochemistry and Centre for Prions and Protein Folding Diseases (CPPFD), University of Alberta, Edmonton, AB T6G 2E1, Canada

关键词: eIF4B;    p70S6K;    Akt;    Multiplex Western blots;    Protein kinase;    Kinomics;    Neurotoxicity;    Cytoplasmic PrP;    Prion disease;   

Others  :  1148247
DOI  :  10.1186/1743-422X-11-175

 received in 2014-06-05, accepted in 2014-09-26,  发布年份 2014

【 摘 要 】

Background

Dysregulated protein kinase signaling is involved in the pathogenesis of many chronic diseases. However, the dysregulated signaling pathways critical to prion pathogenesis remain incompletely characterized. Global analyses of signaling pathways may be useful to better characterize these pathways. We therefore set out to develop such global assays. To this end, we used as a model cytoplasmic mutants of the cellular prion protein (PrP^C), which are toxic to N2a neuroblastoma cells. We tested the global assays for their sensitivity to detect changes in signaling pathways in cells expressing cytoplasmic PrP mutants.

Methods

We developed a targeted proteomics (kinomics) approach using multiplex Western blots to identify signaling pathways dysregulated in chronic neurological pathologies. We tested the approach for its potential ability to detect signaling changes in N2a cells expressing cytoplasmic PrP mutants.

Results

Multiplex Western blots were designed to quantitate the expression levels of 137 protein kinases in a single membrane and using only 1.2 mg of sample. The response of the blots was sensitive and linear to changes of 6% in protein levels. Hierarchical and functional clustering of the relative expression levels identified an mTOR signaling pathway as potentially dysregulated in N2a cells expressing cytoplasmic PrP. The mTOR signaling pathway regulates global protein synthesis, which is inhibited in cells expressing cytoplasmic PrP. The levels of proteins involved in the Akt1/p70S6K branch of mTOR signaling changed in synchrony with time of cytoplasmic PrP expression. Three kinases in this pathway, Akt, p70S6K, and eIF4B were in their inactive states, as evaluated by phosphorylation of their regulatory sites.

Conclusion

The results presented are consistent with the previously reported inhibition of Akt/p70S6K/eIF4B signaling as mediating pathogenesis of cytoplasmic PrP. We conclude that the kinomic analyses are sensitive and specific to detect signaling pathways dysregulated in a simple in vitro model of PrP pathogenesis.

【 授权许可】

   
2014 Shott et al.; licensee BioMed Central Ltd.

【 图 表 】

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