KSHV vFLIP binds to IKK-gamma to activate IKK.
When expressed in heterologous cells, the viral FLIP protein (vFLIP) of Kaposi's-sarcoma-associated herpesvirus (KSHV) has been reported both to block Fas-mediated apoptosis and to activate the NF-kappaB activation pathway by interaction with IkappaB kinase (IKK). In a yeast-two-hybrid screen, we identified IKKgamma as an interacting partner of vFLIP. We expressed fragments of IKKgamma in mammalian cells and bacteria, and identified the central CCR3/4 (amino acids 150-272) as the vFLIP binding region. To investigate the proteins interacting with vFLIP in a KSHV-infected primary effusion lymphoma (PEL) cell line, we immunoprecipitated vFLIP and identified four associated proteins by mass spectrometry: IKK components IKKalpha, beta and gamma, and the chaperone, Hsp90. Using gel filtration chromatography, we demonstrated that a single population of vFLIP in the cytoplasm of PEL cells co-eluted and co-precipitated with an activated IKK complex. An inhibitor of Hsp90, geldanamycin, inhibited IKK's kinase activity induced by vFLIP and killed PEL cells, suggesting that vFLIP activation of IKK contributes to PEL cell survival.
Field
Nigel
N
Department of Immunology and Molecular Pathology, University College London, Windeyer Institute, 46 Cleveland St, London W1T 2AH, UK.
Low
Walter
W
Daniels
Mark
M
Howell
Steven
S
Daviet
Laurent
L
Boshoff
Chris
C
Collins
Mary
M
D017209:Apoptosis / Q000502:physiology; D016227:Benzoquinones; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D047029:Lactams, Macrocyclic; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012514:Sarcoma, Kaposi / Q000378:metabolism / Q000821:virology; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000378:metabolism*
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D016328:NF-kappa B; D021122:Protein Subunits; D011809:Quinones; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C481275:viral FLIP protein, Human herpesvirus 8; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C001277:geldanamycin