TY - JOUR
T1 - Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Requires CADM1/TSLC1 for Inactivation of the NF-κB Inhibitor A20 and Constitutive NF-κB Signaling
AU - Pujari, Rajeshree
AU - Hunte, Richard
AU - Thomas, Remy
AU - van der Weyden, Louise
AU - Rauch, Dan
AU - Ratner, Lee
AU - Nyborg, Jennifer K.
AU - Ramos, Juan Carlos
AU - Takai, Yoshimi
AU - Shembade, Noula
N1 - Funding Information:
The human T-cell lymphocytic cell line Jurkat was obtained from ATCC (Manassas, VA) and Tax-expressing HTLV-1-transformed T-cell lines MT-2, MT-4 and C8166 were obtained from the NIH AIDS Reagent Program. The NEMO-deficient JM4.5.2 cell line [] was a gift from Dr. Sun Sc (The University of Texas MD Anderson, Houston, Texas). Jurkat, JM4.5.2, MT-2, MT-4 and C8166 cells were cultured in RPMI medium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA). Cadm1 and Cadm1 MEFs were generated using a standard procedure []. Briefly, Cadm1 heterozygous mice described previously [] were mated and E12.5 embryos were dissected free of surrounding tissues, washed in PBS (phosphate-buffered saline), and the heads and livers removed. The tissue was placed in 3 ml of 0.25% trypsin/EDTA and disrupted by forcing through a 6 cc syringe followed by vigorous pipetting, and the contents were transferred into a T25 tissue culture flask before placing in a tissue culture incubator at 37 C and 5% CO for 5 minutes. Cadm1 and Cadm1 MEFs were cultured in complete DMEM medium (Mediatech; Manassas, VA) containing 20% fetal bovine serum, heat inactivated, sterile-filtered (Sigma-Aldrich), L-glutamine, 1x penicillin-streptomycin (Invitrogen/Life Technologies). The plasmids pCAGI-Puro-FLAG-CADM1, pCAGI-Puro-FLAG-CADM1-ΔCP (deleting the cytoplasmic tail, aa 404–445), pCAGI-Puro-FLAG-CADM1-ΔEC (deleting the extracellular region, aa 1–362), pCAGI-Puro-FLAG-CADM1-ΔFERM (deleting the FERM domain-binding motif, aa 401–413), and pCAGI-Puro-FLAG- CADM1-ΔPDZ-BM (deleting the PDZ domain-binding motif, aa 442–445) were described previously []. The pCMV4-Tax, Tax M22, Tax M47 and NF-κB-TATA luciferase constructs have been described previously []. Tax M22 and M47 were constructed by replacing G137A and L138S, and L319R and L320S amino acid substitutions using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All mutations were confirmed by DNA sequencing. The following antibodies were used in this study: anti-β-actin (Abcam), anti-TAXBP (Abcam), anti-A20 (BD Biosciences Pharmingen and EMD Millipore), anti-phospho-TAXBP described previously [] was a gift from Dr. Edward Harhaj (Johns Hopkins School of Medicine), anti-ERK1/2, anti-phospho-TAK1, anti-phospho-IκBα, anti-phospho-IKKα/β (Cell Signaling), anti-CADM1 (MBL International Corporation), anti-CADM1, anti-TAK-1, anti-NEMO, anti-IKKα, anti-IKKβ, anti-IκBα (Santa Cruz Biotechnology), anti-Flag (Sigma), anti-NRP (Cayman Chemical), anti-LAT (Upstate Biotechnology), anti-Ubc13 (clone 4E11; Invitrogen), and antibody specific for ubiquitin Lys63 (HWA4C4; Millipore). Anti-Tax [] was prepared from a Tax hybridoma (168B17-46-34) from the AIDS Research and Reference Program of the National Institute of Allergy and Infectious Diseases (US National Institutes of Health). Recombinant TNF and IL-1 were purchased from R&D Systems. The Optiprep was from Axis-Shield (Oslo, Norway). +/+ −/− 0 2 +/+ −/− 1 1 1 1
Publisher Copyright:
© 2015 Pujari et al.
PY - 2015/3/1
Y1 - 2015/3/1
N2 - Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.
AB - Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.
UR - http://www.scopus.com/inward/record.url?scp=84926466755&partnerID=8YFLogxK
U2 - 10.1371/journal.ppat.1004721
DO - 10.1371/journal.ppat.1004721
M3 - Article
C2 - 25774694
AN - SCOPUS:84926466755
VL - 11
SP - 1
EP - 27
JO - PLoS Pathogens
JF - PLoS Pathogens
SN - 1553-7366
IS - 3
M1 - e1004721
ER -