Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

Nicole S Harhaj,Shao-Cong Sun,Edward W Harhaj

doi:10.1074/jbc.m611031200

Nicole S Harhaj, Shao-Cong Sun + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m611031200

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Abstract

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

Highlights

The human T cell leukemia virus type I (HTLV-1)3 is associated with adult T cell leukemia (ATL), an aggressive malignancy of CD4ϩ T cells, and a neuroinflammatory disease known as Human T cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis [1]
We have observed that the perinuclear IKK in Tax-expressing cells colocalizes with the Golgi, and disruption of Golgi with either nocodazole or brefeldin A leads to a redistribution of IKK to the cytoplasm. These results demonstrate that Tax induces relocalization of the IKK complex in a ubiquitin-dependent manner, and dynamic changes in the subcellular localization of the IKK complex may be critical for Tax function
To understand the mechanism of IKK activation by Tax, we examined whether Tax alters the subcellular localization of individual IKK subunits in T lymphocytes

Summary

EXPERIMENTAL PROCEDURES

Antibodies, Plasmids, and Other Reagents—Rabbit anti-IKK␥ (FL419) and mouse anti-IKK␣ (B8) were from Santa Cruz (Santa Cruz, CA). ED40515(Ϫ), MT-1, and TL-OM1 cells are clones of leukemic cells derived from ATL patients, kindly provided by Dr Michiyuki Maeda (Kyoto University) These cells lack Tax expression but exhibit constitutive NF-␬B activation [22]. 293-T cells were transfected with 1 ␮g of pCL-ampho, 0.15 ␮g of VSV-g and either 1 ␮g of pCLXSN, pCLXSN-Tax WT, M22, M47, or K263R,K280R,K284R mutants, or pCLXSN-GFP using FuGENE 6 (Roche Applied Science). Primary antibody incubations were for 1 h, followed by five washes in PBS containing 0.1% Triton X-100. Secondary antibody incubations were for 1 h, followed by three washes in PBS containing 0.1% Triton X-100. EMSA—Nuclear extracts were prepared from transfected 293-T cells as described previously [24]. Reverse Transcription-PCR—Total RNA was isolated from cells infected with empty vector pCLXSN or pCLXSN Tax using the RNeasy Kit (Qiagen). Activation of the reporter by Tax or Tax mutants was calculated as fold induction compared with empty vector

RESULTS

DISCUSSION

Tax physically interacts with the