Novel Hsp90 partners discovered using complementary proteomic approaches
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Hsp90 is an essential eukaryotic molecular chaperone that stabilizes a large set of client proteins, many of which are involved in various cellular signaling pathways. The current list of Hsp90 interactors comprises about 200 proteins and this number is growing steadily. In this paper, we report on the application of three complementary proteomic approaches directed towards identification of novel proteins that interact with Hsp90. These methods are coimmunoprecipitation, pull down with biotinylated geldanamycin, and immobilization of Hsp90β on sepharose. In all, this study led to the identification of 42 proteins, including 18 proteins that had not been previously characterized as Hsp90 interactors. These novel Hsp90 partners not only represent abundant protein species, but several proteins were identified at low levels, among which signaling kinase Cdk3 and putative transcription factor tripartite motif-containing protein 29. Identification of tetratricopeptide-repeat-containing mitochondrial import receptor protein Tom34 suggests the involvement of Hsp90 in the early steps of translocation of mitochondrial preproteins. Taken together, our data expand the knowledge of the Hsp90 interactome and provide a further step in our understanding of the Hsp90 chaperone system.
KeywordsHeat shock protein 90 Identification Immobilization Interactome Kinase Partners
cyclin-dependent kinase 3
S.R. was supported by a Marie-Curie-Excellent Grant of the EU, a VIDI grant of the Netherlands Organization for Science (NWO), and a High Potential Grant of Utrecht University. The authors thank the Netherlands Proteomics Center for financial support. The authors thank Dimitrios Argyropoulos for his assistance in experimental work.
- Millson SH, Truman AW, King V, Prodromou C, Pearl LH, Piper PW (2005) A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in the activity of a stress-activated mitogen-activated protein kinase, Slt2p (Mpk1p). Eukar Cell 4:849–860. doi: 10.1128/EC.4.5.849-860.2005 CrossRefGoogle Scholar
- Modi S, Stopeck AT, Gordon MS, Mendelson D, Solit DB, Bagatell R, Ma W, Wheler J, Rosen N, Norton L, Cropp GF, Johnson RG, Hannah AL, Hudis CA (2007) Combination of trastuzumab and tanespimycin (17-AAG, KOS-953) is safe and active in trastuzumab-refractory HER-2 overexpressing breast cancer: a phase I dose-escalation study. J Clin Oncol 25(Modi, S., Stopeck, A.T., Gordon, M.S., Mendelson, D., Solit, D.B., Bagatell, R., Ma, W., Wheler, J., Rosen, N., Norton, L., Cropp, G.F., Johnson, R.G., Hannah, A.L., and Hudis, C.A):5410–5417. doi: 10.1200/JCO.2007.11.7960 CrossRefPubMedGoogle Scholar
- Pratt WB, Toft DO (2003) Regulation of signaling protein function and trafficking by Hsp90/Hsp70-based chaperone machinery. Exp Biol Med 228:11–133Google Scholar
- Prince T, Sun L, Matts RL (2006) Cdk2: a genuine protein kinase client of Hsp90 and Cdc37. Biochem 44:15287–15295Google Scholar
- Ramalingam SS, Egorin MG, Ramanathan RK, Remick SC, Sikorski RP, Lagattuta TF, Chatta GS, Friedland dM, Stoller RG, Potter DM, Ivy SP, Belani CP (2008) A phase I study of 17-allylamino-17-demethoxygeldanamycin combined with paclitaxel in patients with advanced solid malignancies. Clin Cancer Res 14:3456–3461. doi: 10.1158/1078-0432.CCR-07-5088 CrossRefPubMedGoogle Scholar
- Picard D (2008) Hsp90 interactors. http://www.picard.ch/downloads/Hsp90interactors.pdf
- Zhao R, Davey M, Hsu Y, Kaplanek P, Tong A, Parsons AB, Krogan N, Cagney G, Mai D, Greenblatt J, Boone C, Emili A, Houry WA (2005) Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the Hsp90 chaperone. Cell 120:715–727. doi: 10.1016/j.cell.2004.12.024 CrossRefPubMedGoogle Scholar
- Zhao R, Kakihara Y, Gribun A, Huen J, Yang G, Khanna M, Costanzo M, Brost RL, Boone C, Hughes TR, Yip CM, Houry WA (2008) Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation. J Cell Biol 180:563–578. doi: 10.1083/jcb.200709061 CrossRefPubMedGoogle Scholar