Dr. Edward Harhaj
Associate Professor of Oncology
B.A., M.S. Bucknell University Ph.D., Pennsylvania State College of Medicine
Postdoctoral Fellow, Department of Microbiology and Immunology, Pennsylvania State College of Medicine
Postdoctoral Fellow, Department of Immunobiology, Yale University
Mechanisms of HTLV-1 Tax-mediated NF-κB activation
Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that has been linked to the development of two distinct diseases in humans: 1) a neuroinflammatory disorder termed HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) that is clinically similar to multiple sclerosis (MS), and 2) an aggressive CD4+CD25+ malignancy known as adult T-cell leukemia/lymphoma (ATLL). The NF-κB transcription factor is one of the primary targets of the HTLV-1 encoded oncoprotein Tax to transform cells. Furthermore, NF-κB is required for the survival of HTLV-1 transformed cell lines and ATL leukemic cells. Therefore, we are interested in determining the mechanisms of Tax-mediated NF-κB activation. We have found that Tax is polyubiquitinated in a noncanonical manner whereby the ubiquitin chains are linked via lysine 63 (K63) rather than lysine 48 (K48) that triggers proteosomal degradation. K63-linked polyubiquitination of Tax likely confers a scaffolding function that enables the recruitment of signaling molecules that activate the IαB kinase (IKK) and NF-κB. We have identified the E2 ubiquitin conjugating enzyme Ubc13 as essential for both Tax ubiquitination and NF-κB activation. Future studies will use proteomics, yeast two-hybrid and shRNA screens to identify host factors that regulate Tax ubiquitination and Tax-induced NF-κB activation.
Negative regulation of NF-κB and inflammation
The NF-κB transcription factor is critical for the induction of genes regulating innate and adaptive immunity. NF-κB is tightly regulated to ensure that activation is only transient upon stimulation by cytokines such as tumor necrosis factor alpha (TNF-α and interleukin-1 (IL-1). Dysregulation of NF-κB leading to its persistent activation has been linked to chronic inflammation and autoimmunity. It has also become increasingly clear that chronic inflammation may fuel cancer growth and progression. The zinc finger protein A20 (also known as TNFAIP3) has been shown to be essential for the termination of NF-κB signaling in tumor necrosis factor receptor (TNFR) and Toll-like receptor 4 (TLR4)/IL-1R signaling pathways by targeting key signaling molecules such as RIP1 and TRAF6 for inactivation. Mice lacking A20 die prematurely of multi-organ inflammation and cachexia. In addition, specific polymorphisms within the A20 genomic region have been associated with a number of human autoimmune diseases such as rheumatoid arthritis, systemic lupus erythomatosus and Crohn’s disease. Additional studies have demonstrated loss-of-function mutations or deletions within A20 that are associated with B-cell lymphomas. Despite the importance of A20 in limiting inflammatory responses and tumor growth, little is known about how A20 is regulated and also if other molecules are essential for the termination of NF-κB signaling. Our laboratory has identified several key regulatory proteins that are required for the function of A20 and the down-regulation of NF-κB signaling. A20, TAX1BP1, Itch and RNF11 together constitute the A20 ubiquitin-editing complex that plays a critical role in the repression of NF-κB signaling and inflammation. In response to stimulation with proinflammatory cytokines, A20 inducibly interacts with TAX1BP1, Itch and RNF11 upon phosphorylation of TAX1BP1 by the IKK subunit, IKKα. In addition, HTLV-1 Tax promotes persistent NF-κB signaling and oncogenesis, in part by blocking TAX1BP1 phosphorylation and inactivating the function of A20. In future studies, we will determine the precise roles of each of the subunits of the A20 ubiquitin-editing complex by performing biochemical studies and gene targeting in mice.
Harhaj, N.S., S.C. Sun and E.W. Harhaj. 2007. Activation of NF-κB by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein is associated with ubiquitin-dependent relocalization of IKK. J. Biol. Chem. 282: 4185-92.
Ramos, J.C., P. Ruiz, Jr., L. Ratner, I.M. Reis, C. Brites, C. Pedreso, G.E. Byrne, Jr., N. K. Toomey, V. Andela, E.W. Harhaj, I. Lossos and W.J. Harrington. 2007. IRF-4 and c-rel expression in antiviral therapy resistant adult T-cell leukemia/lymphoma. Blood. 109: 3060-8.
Harhaj, N.S., B. Janic, J.C. Ramos, W.J. Harrington and E.W. Harhaj. 2007. Deregulated expression of CD40 Ligand in HTLV-I infection: distinct mechanisms of downregulation in HTLV-I transformed cell lines and ATL patients. Virology. 362: 99-108.
Shembade, N, N.S. Harhaj, D.J. Liebl and E.W. Harhaj. 2007. Essential role for TAX1BP1 in the termination of TNF-α, IL-1 and LPS-mediated NF-κB and JNK signaling. EMBO J. 26: 3910-22. [Highlighted in Nat. Immunol. 2007 8: 1039].
Shembade, N, N.S. Harhaj, M. Yamamoto, S. Akira and E.W. Harhaj. 2007. The HTLV-I Tax oncoprotein requires the ubiquitin conjugating enzyme Ubc13 for NF-κB activation. J. Virol. 81: 13735-42.
Shembade, N, N.S. Harhaj, K. Parvatiyar, N.G. Copeland, N.A. Jenkins, L.M. Matesic and E.W. Harhaj. 2008. The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin editing enzyme A20. Nat. Immunol. 9: 254-262. [Related News and Views published in Nat. Immunol 9: 227-9; News and Commentary published in Immunol. Cell Biol. 86: 299-300; Faculty of 1000 citation: "Recommended"].
Shembade, N, K. Parvatiyar, N.S. Harhaj and E.W. Harhaj. 2009. The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signaling. EMBO J. 28: 513-22. [Related "Have you seen…?" article published in EMBO J. 28: 455-6].
Shembade, N, A. Ma and E.W. Harhaj. 2010. Inhibition of NF-κB signaling by A20 through disruption of ubiquitin enzyme complexes. Science. 327: 1135-9 [Highlighted in Nat. Immunol. 2010 11: 287; Faculty of 1000 citation: "Must Read"].
Parvatiyar, K, G.N. Barber and E.W. Harhaj. 2010. TAX1BP1 and A20 inhibit antiviral signaling by targeting TBK1/IKKi kinases. J. Biol. Chem. 285: 14999-15009.
Parvatiyar, K and E.W. Harhaj. 2010. Anchors away for ubiquitin chains. Science. 328: 1244-5.
Harhaj, E.W. and V.M. Dixit. 2011. Deubiquitinases in the regulation of NF-κB signaling. Cell Res. 21: 22-39.
Charoenthongtrakul, S, Q. Zhou, N. Shembade, N.S. Harhaj and E.W. Harhaj. 2011. HTLV-I Tax inhibits antiviral signaling via NF-κB-dependent induction of SOCS1. J. Virol. 85: 6955-6962.
Shembade, N., R. Pujari, N.S. Harhaj, D.W. Abbott and E.W. Harhaj. 2011. The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1. Nat. Immunol. 12: 834-843 [Related News and Views published in Nat. Immunol. 2011 12: 815-16].
Gao, L., H. Coope, S. Grant, A. Ma, S.C Ley and E.W. Harhaj. 2011. ABIN1 cooperates with TAX1BP1 and A20 to inhibit antiviral signaling. J. Biol. Chem. 286: 36592-36602.
Lavorgna, A. and E.W. Harhaj. 2012. An RNAi screen identifies the deubiquitinase STAMBPL1 as a critical regulator of HTLV-1 Tax nuclear export and NF-κB activation. J. Virol. Epub Jan. 18.
Lavorgna, A. and E.W. Harhaj. 2012. EBV LMP1: new and shared pathways to NF-κB activation. PNAS. Epub Feb. 2.