Abstract
HSP90 is a central player in the folding and maturation of many proteins. More than two hundred HSP90 clients have been identified by classical biochemical techniques including important signaling proteins with high relevance to human cancer pathways. HSP90 inhibition has thus become an attractive therapeutic concept and multiple molecules are currently in clinical trials. It is therefore of fundamental biological and medical importance to identify, ideally, all HSP90 clients and HSP90 regulated proteins. To this end, we have taken a global and a chemical proteomic approach in geldanamycin treated cancer cell lines using stable isotope labeling with amino acids in cell culture and quantitative mass spectrometry. We identified >6200 proteins in four different human cell lines and ~1600 proteins showed significant regulation upon drug treatment. Gene ontology and pathway/network analysis revealed common and cell-type specific regulatory effects with strong connections to unfolded protein binding and protein kinase activity. Of the 288 identified protein kinases, 98 were geldanamycin treatment including >50 kinases not formerly known to be regulated by HSP90. Protein turn-over measurements using pulsed stable isotope labeling with amino acids in cell culture showed that protein down-regulation by HSP90 inhibition correlates with protein half-life in many cases. Protein kinases show significantly shorter half lives than other proteins highlighting both challenges and opportunities for HSP90 inhibition in cancer therapy. The proteomic responses of the HSP90 drugs geldanamycin and PU-H71 were highly similar suggesting that both drugs work by similar molecular mechanisms. Using HSP90 immunoprecipitation, we validated several kinases (AXL, DDR1, TRIO) and other signaling proteins (BIRC6, ISG15, FLII), as novel clients of HSP90. Taken together, our study broadly defines the cellular proteome response to HSP90 inhibition and provides a rich resource for further investigation relevant for the treatment of cancer.
Highlights
The protein HSP90 is a evolutionary conserved molecular chaperone that is abundantly and ubiquitously expressed in
Using a combination of stable isotope labeling in cell culture [14], core proteome profiling[15], chemical precipitation of kinases[16], and quantitative mass spectrometry [17], we identified Ͼ6200 proteins of which ϳ1600 proteins showed common as well as cell type specific regulation upon drug treatment
Protein turn-over measurements using pulsed stable isotope labeling with amino acids in cell culture (SILAC)1 [18, 19] showed that, for a significant number of proteins, the rate of HSP90 inhibition induced protein downregulation correlates with protein half-life and that protein kinases have significantly shorter half lives than other proteins with potentially important implications for HSP90 inhibition in cancer therapy
Summary
The protein HSP90 is a evolutionary conserved molecular chaperone that is abundantly and ubiquitously expressed in. HSP90 Regulated Proteome precipitation of HSP90 complexes and chemical precipitation using immobilized HSP90 inhibitors [12] These studies have identified some important new HSP90 clients but generally fail to provide a global view of HSP90 regulated proteome because the attained proteomic depth was very limited and many HSP90 interactions are too transient or of too weak affinity to be purified by these methods. We have profiled the global response of the proteomes and kinomes of the four cancer cell lines K562, Colo205, Cal, and MDAMB231 to the HSP90 inhibitor geldanamycin. Using a combination of stable isotope labeling in cell culture [14], core proteome profiling[15], chemical precipitation of kinases[16], and quantitative mass spectrometry [17], we identified Ͼ6200 proteins of which ϳ1600 proteins showed common as well as cell type specific regulation upon drug treatment. The data demonstrate the value of the global drug profiling approach and provides a rich resource for future investigation in HSP90 dependent biological processes
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