Interaction of CARD6 with RICK and RIP1. (A) Mutated CARD6 expression constructs. Numbers identify the position of the indicated residues in the native human CARD6 protein. In the CARD6(RNR) mutant, three amino acid residues of the CARD essential for CARD–CARD interactions (X) are mutated (L30R, D31N, and L33R). All proteins were expressed as fusion proteins with a C-terminal FLAG-tag except for CARD6-CARD, which contains a C-terminal HA-tag. (B–F) HEK293T cells were transfected with empty vector or the indicated FLAG-tagged CARD6 constructs (Top) plus HA-tagged RICK (B and C) or the HA-tagged RIP1 and RICK constructs indicated at the bottom of D–F. Cell lysates were immunoprecipitated with anti-FLAG Ab and immunoblotted with anti-HA(3F10) Ab (and anti-FLAG Ab, where indicated). Fractions of the input were immunoblotted with anti-FLAG (Top) or anti-HA(3F10) Ab (second from top). In F, immunoprecipitations were subjected to a kinase assay before Western blotting (WB). F Bottom shows an autoradiograph of the WB that was scanned before quantification of the HA- and FLAG-tagged proteins in the pulldown with anti-HA(3F10) and anti-FLAG Abs (third panel from top). Results for each experiment are representative of at least three independent trials. WB, Western blot; IP, immunoprecipitation; IgG-H, IgG heavy chain; *, crossreactive band.

Coexpression of RICK induces translocation of CARD6 to aggresomes in a CARD-dependent manner. COS-7 cells expressing FLAG-tagged WT-CARD6 and HA-tagged RICK (Top) were stained with anti-huCARD6 and anti-HA(3F10) Abs. Cells were counterstained with anti-acetylated tubulin Ab (Right). Arrowheads point to areas of aggresome assembly. COS-7 cells cotransfected with FLAG-tagged WT-CARD6 and HA-tagged ΔCARD-RICK (Middle) or FLAG-tagged ΔCARD-CARD6 and HA-tagged ΔCARD-RICK (Bottom), were stained with anti-huCARD6 and anti-HA(12CA5) Abs. Nuclei were counterstained with DAPI (Right). Images showing unmerged counterstains have been excluded.

CARD6 is a general modulator of NF-κB activation pathways. (A) RNA interference-mediated suppression of CARD6. Huh7 cells were electroporated with nonsilencing control siRNA or the indicated CARD6-specific siRNAs. At 48 h after electroporation, the relative amounts of CARD6 RNA were determined by using semiquantitative RT-PCR (Top). Protein extracts (50 μg) were immunoblotted with anti-hu-CARD6 (Middle) or anti-β-actin Abs (Bottom). (B) Reduction of NF-κB activation by suppression of CARD6. Huh7 cells were electroporated with nonsilencing control siRNA or siRNA2 (Upper) or control siRNA or siRNA1 (Lower) and transfected 24 h later with empty vector, TLR4/MD-2, TLR9, NOD1, or BCL10 expression constructs in the presence of the pBVIx-Luc and pRL-TK reporters. Triplicate cultures were treated for 4 h with TNF-α, IL-1β, LPS, or hCpG, as indicated, and NF-κB activation was determined as for Fig. 4A. Results shown are the means of normalized values ± SD of triplicate cultures. Results were normalized to values obtained by cotransfection of reporter constructs with empty vectors (1-fold) after electroporation of control siRNA. (C) Proposed model for CARD6 interactions and function. The PRR of CARD6 is required for its association with the microtubule network. During transient interactions at the microtubules, RICK targets CARD6 to the aggresome in a CARD-dependent manner. Conversely, the CARD6-CARD negatively regulates nonCARD-mediated interactions of CARD6 and RICK. Functionally, CARD6 synergistically enhances NF-κB activation by multiple stimuli via a mechanism that likely requires additional signaling components. Black arrows indicate physical interactions; green arrows and the red connector show positive or negative regulatory mechanisms, respectively; and blue arrows illustrate translocation to the aggresome. (Note: RICK is drawn in reverse orientation.)

CARD6 colocalizes with microtubules in a PRR- and CARD-dependent manner. COS-7 cells expressing FLAG-tagged WT-CARD6 (Left), ΔPRR-CARD6 (Center), or ΔCARD-CARD6 (Right) were stained with anti-huCARD6 and anti-acetylated tubulin Abs. Arrowheads point to specific structures to show colocalization of WT-CARD6 with microtubules (Left). For all immunohistochemical figures, primary Abs were visualized as described in Supporting Text.

CARD6 synergistically promotes NF-κB activation. (A and B) HEK293T cells were transfected with control plasmid or FLAG-tagged human WT-CARD6 and treated with TNFα (A) or cotransfected with HA-tagged NOD1 (A), or cotransfected with RICK, RIP1, BCL10 or mitogen-activated protein kinase kinase kinase 3 (B). Induction of NF-κB activation was determined by using the pBVIx-Luc and pRL-TK luciferase reporter constructs. (C and D) Cotransfections were with the indicated FLAG-tagged human CARD6 variants plus the indicated amounts of HA-tagged RICK. Induction of NF-κB activation was determined as for A and B. Because coexpression of ΔCARD-CARD6 leads to an increase in RICK expression (although other variants inhibit expression), the amounts of transfected RICK cDNA were titrated to provide control samples with similar expression levels. Results shown are the means of normalized values ± SD of triplicate cultures. For A, C, and D, results were normalized to values obtained by cotransfection of reporter constructs with empty vectors (1-fold). For A–D, expression of transfected constructs was determined by Western blotting with anti-FLAG and anti-HA(3F10) Abs (Top). The amount of extract subjected to SDS/PAGE in each case was normalized by Renilla luciferase (pRL-TK) assay.