Tax requires CADM1 for NF-κB activation.

<p>(A) Lentiviral Tax was transduced in Jurkat T-cells stably expressing control scrambled shRNA or CADM1 shRNA. After 48 h, lysates were subjected to immunoblotting with anti-IκBα, anti-phospho-IκBα, anti-CADM1, anti-Tax, and anti-β-actin antibodies. (B) Primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were transduced with Tax-expressing lentiviruses. After 48 h, lysates were subjected to immunoblotting with anti-IκBα, anti-phospho-IκBα, anti-CADM1, anti-Tax, and anti-β-actin antibodies. (C) Nuclear extracts from lentiviral expressing Tax in primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were used for NF-κB and Oct-1 EMSA, and cytoplasmic extract were subjected to immunoblotting with anti-Tax, anti-CADM1, and anti-β-actin antibodies. (D) Lysates from HTLV-1 transformed C8166, MT-2, and MT-4 cells stably expressing control scrambled shRNA or CADM1 shRNA were subjected to immunoblotting with anti-IκBα, anti-phospho-IκBα, anti-CADM1, anti-Tax, and anti-β-actin antibodies. (E) Nuclear extracts from HTLV-1 transformed C8166, MT-2, and MT-4 cells stably expressing control scrambled shRNA or CADM1 shRNA were used for NF-κB and Oct-1 EMSA, and cytoplasmic extracts were subjected to immunoblotting with anti-CADM1 and anti-β-actin antibodies. (F) Primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were transduced with Tax-expressing lentiviruses as described for panel B. After 48 hours, RNA was prepared and subjected to RT-PCR for A20, IL-6, Bfl-1, Tax, and GAPDH expression.</p

Tax interacts with CADM1.

<p>(A) Jurkat T-cells were transduced with Tax-expressing lentiviruses. After 48 hours, cells were lysed and immunoprecipitated with either anti-Tax or control anti-IgG, followed by immunoblotting with anti-Tax and anti-CADM1 antibodies. Lysates were examined for Tax, CADM1, and β-actin expression. (B) Lysates from Tax expressing HTLV-1 transformed C8166, MT-2 and MT-4 cells were immunoprecipitated in duplicate with either anti-CADM1 or control anti-IgG, followed by immunoblotting with anti-Tax and anti-CADM1. Lysates were examined for Tax, CADM1, and β-actin expression. (C) A schematic overview of the FLAG-CADM1 deletion mutants ΔSP, ΔCP, ΔEC, ΔPDZ-BM and ΔFERM. (D) Mapping the interaction between CADM1 and Tax. Primary <i>Cadm1</i>^{<i>−/−</i>} MEFs were transfected with Tax expression vector with the indicated Flag-CADM1 mutants. After Thirty-six hours post-transfection, proteins from lysates were immunoprecipitated with anti-Flag and detected by immunoblotting with anti-Tax and anti-Flag antibodies. Lysates were immunoblotted with anti-Tax, and anti-Flag antibodies.</p

Model of the role of CADM1 in Tax-mediated NF-κB activation.

<p>Membrane associated CADM1 recruits Ubc13 on Tax, which causes K63-linked polyubiquitination of Tax, and association of TAX₁BP_1, NRP, and the NEMO/IKK complex with Tax. CADM1 also inhibits IKKα-mediated phosphorylation of TAX₁BP₁ and disrupts the NF-κB negative regulator ubiquitin-editing enzyme A20 complex assembly that allows chronic NF-κB activation in HTLV-1 transformed cells.</p

Tax induces CADM1 expression.

<p>(A) CADM1 expression in foot, tail, spleen and bone marrow (BM) tissues derived from spontaneous tumors of 14 month old control Tax-negative and Tax-positive transgenic mice. Immunoblotting was performed with anti-CADM1, Tax, and β-actin antibodies. CADM1 expression in lentiviral-transduced empty vector wildtype Tax or Tax mutants (M22 or M47) in primary MEFs (B) and Jurkat T-cells (C) was analyzed with anti-CADM1, SOCS1, Tax, and β-actin antibodies.</p

Membrane associated CADM1 mediates K63-linked polyubiquitination of Tax and links Tax adaptor proteins in the lipid rafts.

<p>(A) MT-2 cells were stained with DAPI, anti-Tax, anti-CADM1, and anti-GM-130, and subjected to confocal microscopy. (B) MT-2 cells were stained with DAPI, anti-Tax, anti-CADM1, and cholera toxin B conjugated with red fluorescence to detect GM-1, and subjected to confocal microscopy. (C) Lipid raft fractionations from MT-2 cells stably expressing control scrambled shRNA or CADM1 shRNA were split into half and subjected to immunoprecipitation with either anti-Tax or anti-CADM1. Samples immunoprecipitated with anti-Tax were immunoblotted with anti-K63-ubi and anti-Tax. Samples immunoprecipitated with anti-CADM1 were immunoblotted with anti-CADM1, anti-TAX₁BP₁, anti-Tax, anti-NEMO, anti-Ubc13, and anti-NRP antibodies. Lysates from lipid rafts fractions were examined for Tax, phospho-IKKα/β, total IKKα, IKKβ, NEMO, CADM1, Ubc13, TAX₁BP₁, NRP, ERK1 (marker for soluble fractions), LAT (lipid raft protein marker) and GM1 (lipid raft marker).</p

CADM1 is required for Tax K63-linked polyubiquitination.

<p>(A) Lentiviral Tax was transduced in Jurkat T-cells stably expressing control scrambled shRNA or CADM1 shRNA. After 48 hours, cells were lysed and immunoprecipitated with anti-Tax, followed by immunoblotting with anti-Ubi-K63 and anti-Tax antibodies. Lysates were examined for Tax, CADM1 and β-actin expression. (B) Lentiviral Tax was transduced in <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs. After 48 hours cells were lysed and immunoprecipitated with anti-Tax followed by immunoblotting with anti-K63-ubi and anti-Tax antibodies. Lysates were examined for Tax, CADM1, and β-actin expression. (C) Lysates from HTLV-1 transformed (C8166, MT-2, and MT-4) cells stably expressing CADM1 shRNA were immunoprecipitated with anti-Tax followed by immunoblotting with anti-K63-ubi and anti-Tax antibodies. Lysates were examined for Tax, CADM1, and β-actin expression.</p