Abstract
Inhibitor of apoptosis proteins (IAP) are evolutionarily conserved anti-apoptotic regulators1,2. C-RAF protein kinase is a direct RAS effector protein, which initiates the classical mitogen-activated protein kinase (MAPK) cascade. This signalling cascade mediates diverse biological functions, such as cell growth, proliferation, migration, differentiation and survival3,4. Here we demonstrate that XIAP and c-IAPs bind directly to C-RAF kinase and that siRNA-mediated silencing of XIAP and c-IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitylation of C-RAF in vivo through the Hsp90-mediated quality control system, independently of its E3 ligase activity. In addition, XIAP or c-IAP-1/2 knockdown cells showed enhanced cell migration in a C-RAF-dependent manner. XIAP promotes binding of CHIP (carboxy terminal Hsc70-interacting protein), a chaperone-associated ubiquitin ligase, to the C-RAF–Hsp90 complex in vivo. Interfering with CHIP expression resulted in stabilization of C-RAF and enhanced cell migration, as observed in XIAP knockdown cells. Our data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signalling pathway and cell migration.
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Change history
24 November 2008
In the version of this article initially published online, the label c-IAP-1 in Figure 1d was duplicated for both of the lower lines. The omitted c-IAP-2 label has been replaced in the HTML and PDF versions of the article.
References
Eckelman, B. P., Salvesen, G. S., & Scott, F. L. Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family. EMBO Rep. 7, 988–994 (2006).
Salvesen, G. S. & Duckett, C. S. IAP proteins: blocking the road to death's door. Nature Rev. Mol. Cell Biol. 3, 401–410 (2002).
Rajalingam, K., Schreck, R., Rapp, U. R., & Albert, S. Ras oncogenes and their downstream targets. Biochim. Biophys. Acta 1773, 1177–1195 (2007).
Wellbrock, C., Karasarides, M., & Marais, R. The RAF proteins take centre stage. Nature Rev. Mol. Cell Biol. 5, 875–885 (2004).
Srinivasula, S. M. & Ashwell, J. D. IAPs: What's in a name? Mol. Cell 30, 123–135 (2008).
Wright, C. W. & Duckett, C. S. Reawakening the cellular death program in neoplasia through the therapeutic blockade of IAP function. J. Clin. Invest. 115, 2673–2678 (2005).
Vaux, D. L. & Silke, J. IAPs, RINGs and ubiquitylation. Nature Rev. Mol. Cell Biol. 6, 287–297 (2005).
Burstein, E. et al. A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J. 23, 244–254 (2004).
Olayioye, M. A. et al. XIAP-deficiency leads to delayed lobuloalveolar development in the mammary gland. Cell Death Differ. 12, 87–90 (2004).
Alavi, A., Hood, J. D., Frausto, R., Stupack, D. G., & Cheresh, D. A. Role of Raf in vascular protection from distinct apoptotic stimuli. Science 301, 94–96 (2003).
Dhillon, A. S., Hagan, S., Rath, O., & Kolch, W. MAP kinase signalling pathways in cancer. Oncogene 26, 3279–3290 (2007).
Rapp, U. R., Rennefahrt, U., & Troppmair, J. Bcl-2 proteins: master switches at the intersection of death signaling and the survival control by Raf kinases. Biochim. Biophys. Acta 1644, 149–158 (2004).
Tian, S. et al. Interaction and stabilization of X-linked inhibitor of apoptosis by Raf-1 protein kinase. Int. J. Oncol. 29, 861–867 (2006).
Klemke, R. L. et al. Regulation of cell motility by mitogen-activated protein kinase. J. Cell Biol. 137, 481–492 (1997).
Rajalingam, K. et al. Prohibitin is required for Ras-induced Raf–MEK–ERK activation and epithelial cell migration. Nature Cell Biol. 7, 837–843 (2005).
Srinivasula, S. M. et al. A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature 410, 112–116 (2001).
Vucic, D. Targeting IAP (inhibitor of apoptosis) proteins for therapeutic intervention in tumors. Curr. Cancer Drug Targets 8, 110–117 (2008).
da Rocha, D. S. et al. Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer Res. 65, 10686–10691 (2005).
Grbovic, O. M. et al. V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc. Natl Acad. Sci. USA 103, 57–62 (2006).
McDonough, H. & Patterson, C. CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperones 8, 303–308 (2003).
Arndt, V., Rogon, C., & Hohfeld, J. To be, or not to be — molecular chaperones in protein degradation. Cell Mol. Life Sci. 64, 2525–2541 (2007).
Isaacs, J. S., Xu, W., & Neckers, L. Heat shock protein 90 as a molecular target for cancer therapeutics. Cancer Cell 3, 213–217 (2003).
Schulte, T. W. et al. Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf–1–MEK-mitogen-activated protein kinase signalling pathway. Mol. Cell Biol. 16, 5839–5845 (1996).
Schulte, T. W., An, W. G., & Neckers, L. M. Geldanamycin-induced destabilization of Raf-1 involves the proteasome. Biochem. Biophys. Res. Commun. 239, 655–659 (1997).
Schneider, C. et al. Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc. Natl Acad. Sci. USA 93, 14536–14541 (1996).
Young, J. C., Agashe, V. R., Siegers, K., & Hartl, F. U. Pathways of chaperone-mediated protein folding in the cytosol. Nature Rev. Mol. Cell Biol. 5, 781–791 (2004).
Demand, J., Alberti, S., Patterson, C., & Hohfeld, J. Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling. Curr. Biol. 11, 1569–1577 (2001).
Noble, C. et al. CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation. Mol. Cell 31, 862–872 (2008).
Harlin, H., Reffey, S. B., Duckett, C. S., Lindsten, T., & Thompson, C. B. Characterization of XIAP-deficient mice. Mol. Cell Biol. 21, 3604–3608 (2001).
Rajalingam, K. et al. IAP–IAP complexes required for apoptosis resistance of C. trachomatis-infected cells. PLoS Pathog. 2, e114 (2006).
Downward, J. Targeting RAS signalling pathways in cancer therapy. Nature Rev. Cancer 3, 11–22 (2003).
Acknowledgements
We thank Pascal Meier, Richard Marais, Len Neckers, Yuri Lazebnik and H.Wayant for valuable reagents; Brigitte Ruester and Reinhard Henschler for assistance with cell sorting; Renate Metz for help with the purification of proteins; Andreas Fischer for Biacore analysis; Juliane Mooz and Barbara Bauer for technical assistance. This work is supported by an Emmy Noether programme grant (RA1739/1–1) to K.R. from the DFG and various grants to U.R.R. from DFG, including Immunomodulation and German-French Graduate Schools, SFB487, SFB581, and grants from the Scheel Stiftung. E.S.A. is supported by grants from the NIH (GMO76167). We thank Antoine Galmiche for sharing unpublished observations, Ralf Schreck and Ritva Tikkanen for critically reading the manuscript.
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T.D. performed most of the experiments; T.K.O. performed additional experiments; G.S.H. performed the microscopic analyses; C.K. wrote the algorithms and performed the quantification of transwell migration experiments; M.H. performed the BIAcore measurements; E.S.A. provided valuable materials and advice; U.R.R. initiated the project, provided valuable materials and critical advice; K.R. conceived and designed the experiments, analysed and interpreted data with all authors, coordinated the study and wrote the paper.
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Dogan, T., Harms, G., Hekman, M. et al. X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility. Nat Cell Biol 10, 1447–1455 (2008). https://doi.org/10.1038/ncb1804
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DOI: https://doi.org/10.1038/ncb1804