The NF-κB pathway is constitutively activated in adult T cell leukemia, an aggressive malignancy caused by Human T Leukemia Virus type 1 (HTLV-1). The viral oncoprotein Tax triggers this constitutive activation by interacting with the ubiquitin-rich IKK complex. We previously demonstrated that Optineurin and TAX1BP1, two members of the ubiquitin-binding, Sequestosome-1 (SQSTM-1/p62)-like selective autophagy receptor family, are involved in Tax-mediated NF-κB signaling. Here, using a proximity-dependent biotinylation approach (BioID), we identify p62 as a new candidate partner of Tax and confirm the interaction in infected T cells. We then demonstrate that p62 knock-out in MEF cells as well as p62 knock-down in HEK293T cells significantly reduces Tax-mediated NF-κB activity. We further show that although p62 knock-down does not alter NF-κB activation in Jurkat T cells nor in infected T cells, p62 does potentiate Tax-mediated NF-κB activity upon over-expression in Jurkat T cells. We next show that p62 associates with the Tax/IKK signalosome in cells, and identify the 170–206 domain of p62 as sufficient for the direct, ubiquitin-independent interaction with Tax. However, we observe that this domain is dispensable for modulating Tax activity in cells, and functional analysis of p62 mutants indicates that p62 could potentiate Tax activity in cells by facilitating the association of ubiquitin chains with the Tax/IKK signalosome. Altogether, our results identify p62 as a new ubiquitin-dependent modulator of Tax activity on NF-κB, further highlighting the importance of ubiquitin in the signaling activity of the viral Tax oncoprotein.
Since the identification of the antisense protein of HTLV-2 (APH-2) and the demonstration that APH-2 mRNA is expressed in vivo in most HTLV-2 carriers, much effort has been dedicated to the elucidation of similarities and/or differences between APH-2 and HBZ, the antisense protein of HTLV-1. Similar to HBZ, APH-2 negatively regulates HTLV-2 transcription. However, it does not promote cell proliferation. In contrast to HBZ, APH-2 half-life is very short. Here, we show that APH-2 is addressed to PML nuclear bodies in T-cells, as well as in different cell types. Covalent SUMOylation of APH-2 is readily detected, indicating that APH-2 might be addressed to the PML nuclear bodies in a SUMO-dependent manner. We further show that silencing of PML increases expression of APH-2, while expression of HBZ is unaffected. On the other hand, SUMO-1 overexpression leads to a specific loss of APH-2 expression that is restored upon proteasome inhibition. Furthermore, the carboxy-terminal LAGLL motif of APH-2 is responsible for both the targeting of the protein to PML nuclear bodies and its short half-life. Taken together, these observations indicate that natural APH-2 targeting to PML nuclear bodies induces proteasomal degradation of the viral protein in a SUMO-dependent manner. Hence, this study deciphers the molecular and cellular bases of APH-2 short half-life in comparison to HBZ and highlights key differences in the post-translational mechanisms that control the expression of both proteins.
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