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First published online 30 July 2003
doi: 10.1242/jcs.00691

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KSHV vFLIP binds to IKK- to activate IKK

Nigel Field¹, Walter Low^1,*, Mark Daniels¹, Steven Howell², Laurent Daviet³, Chris Boshoff⁴ and Mary Collins^1,

¹ Department of Immunology and Molecular Pathology, University College London, Windeyer Institute, 46 Cleveland St, London W1T 2AH, UK
² National Institute for Medical Research, Laboratory of Protein Structure, The Ridgeway, Mill Hill, London NW7 1AA, UK
³ Hybrigenics, 3-5 Impasse Reille, 75014 Paris, France
⁴ Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK

View larger version (21K): [in a new window]   Fig. 1. vFLIP interacts directly with the IKK subunit of the IKK complex. A human placental cDNA library was screened for proteins interacting with a vFLIP bait using yeast-two-hybrid technology. (A) A schematic representation of IKK with a list of the six independent fusions of IKK (B). Black boxes indicate coiled-coil regions (CCR), white boxes indicate the leucine zipper domains (LZ) essential for interaction of HTLV-1 Tax with IKK and the grey box indicates a zinc finger motif (ZF). The N-terminus of IKK is responsible for interaction with IKK and ß, whereas the C-terminus is required for activation of the IKK complex. Truncation mutants of IKK, generated as Xpress tag or GST fusions, are shown (C). (D) The interaction of Xpress tagged IKK truncation mutants with vFLIP when both are overexpressed in 293T cells. Cell lysates were immunoprecipitated using an anti-vFLIP antibody and analysed by immunoblot probed with an anti-Xpress antibody. (E) Interaction of vFLIP transiently expressed in 293T cells with GST fusion IKK mutants.

View larger version (40K): [in a new window]   Fig. 2. Identification of proteins interacting with vFLIP in a KSHV-infected B-cell line. Proteins were immunoprecipitated from the KSHV-infected PEL cell line BC3 using a rat IgG control or the anti-vFLIP monoclonal antibody 6/14 and then run on a 12% SDS-polyacrylamide gel. (A) Silver stain of tracks containing 10% of the immunoprecipitated protein. (B) Colloidal Coomassie stain of part of a parallel pair of tracks containing 90% of the immunoprecipitated protein. Bands detected in the vFLIP immunoprecipitate, but not the control, were excised from the Coomassie stained gel and identified by mass spectrometry as indicated.

View larger version (36K): [in a new window]   Fig. 3. Analysis of the vFLIP-IKK complex in a KSHV-infected B-cell line. (A) Cytoplasmic extract from BC3 cells was subjected to gel filtration on a Superose 6 column, then fractions were analysed by immunoblot with anti-IKK, ß or , anti-vFLIP and anti-vCyclin antibodies. Fractions were also immunoprecipitated with the anti-vFLIP antibody 6/14 and IB kinase activity was measured. The elution volume of protein standards is indicated. (B) Cytoplasmic extracts from BC3 cells were immunoprecipitated using the anti-vFLIP antibody or an isotype-matched control. The immune complexes were incubated with wild-type or mutant (S32A/S36A) GST-IB substrates. The lower panel shows a Coomassie stained gel of the GST-IB in each reaction. (C) Cytoplasmic extract from BC3 cells was immunoprecipitated using an anti-IKK antibody. Equivalent proportions of the original extract (Total lysate), the supernatant from the immunoprecipitation (Supernatant) and the immunoprecipitate (IKKIP) were then analysed by immunoblot with anti-vFLIP and anti-IB antibodies.

View larger version (30K): [in a new window]   Fig. 4. The vFLIP-IKK kinase activity is inhibited by geldanamycin. Cytoplasmic extract from 293T cells transduced with a lentivirus encoding either vFLIP plus GFP (A,B) or GFP alone (C) and then treated with DMSO or geldanamycin (GA), as indicated, was subjected to gel filtration on a Superose 6 column. Fractions were analysed by immunoblot with anti-vFLIP or anti-IKK antibodies; in A and B fractions were also immunoprecipitated with the anti-vFLIP antibody 6/14 and IB kinase activity was measured.

View larger version (24K): [in a new window]   Fig. 5. BC3 cells are killed by geldanamycin (GA). (A) Cytoplasmic extracts from BC3 cells treated with DMSO or GA were immunoprecipitated using the anti-vFLIP antibody and their GST-IB kinase activity measured. The viability of BC3 cells treated with increasing concentrations of GA or equivalent DMSO was evaluated by haemocytometer (B) and by propidium iodide (PI)/annexin V stain followed by FACScan analysis (C) at 48 hours. In each of the four graphs in C, the lower left quadrant represents live unstained cells, the lower right quadrant represents single stained early apoptotic cells, the upper right represents double stained, late apoptotic cells, and the upper left represents necrotic cells. (D) Graphical representation of the data in C.

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