Disruption of Hsp90 Function Results in Degradation of the Death Domain Kinase, Receptor-interacting Protein (RIP), and Blockage of Tumor Necrosis Factor-induced Nuclear Factor-κB Activation

The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor κB (NF-κB) activation. The activation of NF-κB protects cells against TNF-induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. Here we report that RIP is a novel Hsp90-associated kinase and that disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IκB kinase and NF-κB activation. MG-132, a specific proteasome inhibitor, abrogated GA-induced degradation of RIP but failed to restore the activation of IκB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulated in a detergent-insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-κB activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-κB activation by GA may be a critical component of the anti-tumor activity of this drug. The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor κB (NF-κB) activation. The activation of NF-κB protects cells against TNF-induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. Here we report that RIP is a novel Hsp90-associated kinase and that disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IκB kinase and NF-κB activation. MG-132, a specific proteasome inhibitor, abrogated GA-induced degradation of RIP but failed to restore the activation of IκB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulated in a detergent-insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-κB activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-κB activation by GA may be a critical component of the anti-tumor activity of this drug. tumor necrosis factor TNF receptor nuclear factor κB c-Jun N-terminal kinase TNFR-associated factor 2 receptor-interacting protein IκB kinase heat shock protein geldanamycin geldampicin glutathioneS-transferase hemagglutinin phosphate-buffered saline cycloheximide Tumor necrosis factor (TNF)1 is a proinflammatory cytokine that plays a critical role in diverse cellular events, including cell proliferation, differentiation, and apoptosis (1.Tracey K.J. Cerami A. Annu. Rev. Cell Biol. 1993; 9: 317-343Crossref Cell Biol. of TNF-mediated be by one of the TNF and of to the factor receptor apoptosis is and this receptor is also a death receptor a death domain 1993; Biol. 1993; TNF-induced activation of factor NF-κB and c-Jun N-terminal kinase of apoptosis are The that is that the of TNF to to the of and the of the death domain protein the receptor complex a to other proteins the death domain protein TNFR-associated factor 2 and the receptor interacting protein (RIP), to Biol. is essential TNF-induced the of RIP and are activation of NF-κB and A. A. A. A. A. A. The role of RIP in TNF-induced NF-κB activation in an that NF-κB activation protects cells TNF-induced cells are to apoptosis following TNF factor NF-κB is of of Annu. Rev. Cell Biol. NF-κB is to the its proteins its nuclear the proinflammatory TNF and are by IκB kinase in the complex and of this complex A. The degradation of to the of NF-κB and NF-κB to the its a of and NF-κB activation protects cells of apoptosis and cell and NF-κB activity in of shock proteins (Hsps) are a of chaperone proteins that help to protein stability and to degradation proteins cells are to heat shock other Hsp90 is one of the most abundant and stability and function of a of this chaperone is involved in a of important including cell and Hsp90 a specific of the novel anti-tumor geldanamycin A. Biol. 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Cell Biol. 1993; 9: 317-343Crossref Cell Biol. of TNF-mediated be by one of the TNF and of to the factor receptor apoptosis is and this receptor is also a death receptor a death domain 1993; Biol. 1993; TNF-induced activation of factor NF-κB and c-Jun N-terminal kinase of apoptosis are The that is that the of TNF to to the of and the of the death domain protein the receptor complex a to other proteins the death domain protein TNFR-associated factor 2 and the receptor interacting protein (RIP), to Biol. is essential TNF-induced the of RIP and are activation of NF-κB and A. A. A. A. A. A. The role of RIP in TNF-induced NF-κB activation in an that NF-κB activation protects cells TNF-induced cells are to apoptosis following TNF factor NF-κB is of of Annu. Rev. Cell Biol. NF-κB is to the its proteins its nuclear the proinflammatory TNF and are by IκB kinase in the complex and of this complex A. 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