In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax.
Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.
Mukherjee
Sohini
S
Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Negi
Veera S
VS
Keitany
Gladys
G
Tanaka
Yuetsu
Y
Orth
Kim
K
D000107:Acetylation / Q000187:drug effects; D001426:Bacterial Proteins / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D016356:Gene Products, tax; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D016356:Gene Products, tax; D018841:HSP90 Heat-Shock Proteins; D046912:Multiprotein Complexes; D016328:NF-kappa B; D011994:Recombinant Proteins; C109227:YopP protein, Yersinia; D051550:I-kappa B Kinase; D010749:Phosphoprotein Phosphatases