Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax⅐Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased

The human T-cell leukemia virus type 1 oncoprotein Tax is a phosphoprotein with a predominately nuclear subcellular localization that accomplishes multiple functions via protein-protein interactions. It has been proposed that regulation of this protein's pleiotropic functions may be accomplished through phosphorylation of specific amino acid residues. We have conducted a phosphoryl mapping of mammalian-expressed Tax protein using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved physical coverage of 77% of the Tax sequence and identified four novel sites of phosphorylation at Thr-48, Thr-184, Thr-215, and Ser-336. Previously identified potential serine phosphorylation sites at Ser-10, Ser-77, and Ser-274 could not be confirmed by mass spectrometry. The functional significance of these novel phosphorylation events was evaluated by mutational analysis and subsequent evaluation for activity via both CREB and NF-B-responsive promoters. Our results demonstrate that phosphorylation at Thr-215 is associated with loss of both Tax functions, phosphorylation at Thr-48 was specifically deficient for activation via NF-B, and phosphorylation at Thr-184 and Ser-336 had no effect on these Tax functions. Semiquantitation of phosphopeptides revealed that the majority of Tax was phosphorylated at Thr-48, Thr-184, Thr-215, and Ser-336, whereas only a minor population of Tax was phosphorylated at either Ser-300 or Ser-301. These results suggest that both positive and negative phosphorylation signals result in the maintenance of a subfraction of Tax as "active" protein. Human T-cell leukemia virus type 1 (HTLV-1)2 is a human transforming retrovirus. Infection with HTLV-1 can give rise to adult T-cell leukemia and HTLV-1-associated myelopathy/ tropical spastic paraparesis as well as other subneoplastic conditions (1-5). Although cellular transformation can be achieved by expression of a single viral transactivating protein, Tax, the exact mechanism of transformation is not known (6). Tax is thought to induce genomic instability and, thus, to contribute to cellular transformation through interaction with cellular proteins involved in cell cycle control and the DNA damage repair response (7-11). In addition, Tax can activate or repress a variety of cellular genes predominately through the CREB (cAMP-response element-binding protein) and NF-B pathways (6, 12). Thus, uncovering the regulatory mechanism for controlling the various Tax activities is critical to understanding HTLV-1-mediated cellular transformation.There have been several structure-function studies of the Tax protein predominately utilizing molecular biology techniques. Specifically, with respect to the regulation of multiple Tax activities, it has been noted that mutation or deletion of individual domains results in a selective loss of function (13). Important domains for Tax function include a nuclear localization signal (13, 14), nuclear export signal (15, 16), activatio...

Infection with human T-cell leukemia virus induces cellular genomic instability mediated through the viral oncoprotein Tax. Here we present evidence that Tax undermines the cellular DNA damage response by sequestration of damage response factors. We show by confocal microscopy that Tax forms damage-independent nuclear foci that contain DNA-PK, BRCA1, and MDC1. Tax sequesters MDC1 to chromatin sites distinct from classic ionizing radiation-induced foci. The recruitment of MDC1 is competitive between the two foci. The N-terminal region of Tax is sufficient for foci localization, and the C-terminal half is critical for binding to MDC1 and recruitment of additional response factors. Tax expression and DNA damage response factor recruitment repressed the formation of ionizing radiation-induced Nbs1-containing foci. The Tax-induced “pseudo” DNA damage response results in phosphorylation and monoubiquitylation of H2AX, which is ablated by siRNA suppression of MDC1. These data support a model for virus-induced genomic instability in which viral oncogene-induced damage-independent foci compete with normal cellular DNA damage response.

The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)-modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax IntroductionHuman T-cell Leukemia Virus Type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia. 1 Immortalization and transformation of T lymphocytes can be attributed to the expression and activity of the viral oncoprotein Tax. 2 Although the exact mechanism of Tax-mediated transformation is unknown, studies indicate that Tax expression results in genomic instability via chronic disruption of the cellular DNA damage response. 3 It is generally accepted that cellular transformation is a by-product of the long period of genomic instability.Tax exhibits pleiotropic functionality, which is at least partly regulated by subcellular localization to nuclear or cytoplasmic compartments. 3,4 We demonstrated previously that Tax shuttles between the nucleus and cytoplasm. 5 The mechanism for the regulation of Tax localization is unknown, although localizationspecific structural elements have been uncovered. The Tax protein contains a nuclear localization signal, 6 and we recently identified a specific signal sequence that targets Tax to nuclear bodies. 7 We and others have described a nuclear export signal. 5,8 In addition, Lamsoul et al showed that ubiquitylation of Tax is correlated with its accumulation in the cytoplasm, whereas sumoylation of Tax is required for nuclear localization. 9 Therefore, the molecular switch for nuclear versus cytoplasmic localization of Tax at least in part depends on the protein modifiers Small Ubiquitin-like Modifier (SUMO) and ubiquitin, but the mechanism underlying this switch is unknown.Coincident sumoylation and ubiquitylation of a substrate protein is common, and in some cases, these modifications work cooperatively to regulate specific biologic processes (for review, see Ulrich 10 ). The recent discovery of a novel class of Really Interesting New Gene (RING)-domain proteins called SUMOtargeted ubiquitin ligases (STUbLs) has contributed to our understanding of how ubiquitylation of proteins is regulated. STUbL proteins contain SUMO-interacting motifs (SIMs) to interact with the SUMO or SUMO-like domains of their ubiquitylation targets. Therefore, STUbLs are well suited to play an important role in the cross-talk between SUMO and ubiquitin pathways. 11 The role of STUbLs was first realized by studying 2 RING domain-containing proteins, Slx5 and Slx8, in budding and fission yeast. [12][13][14][15] The Slx5/Slx8 complex was subsequently found to mediate quality control of...

The human T-cell leukemia virus type 1 oncoprotein Tax has pleiotropic activities, a subset of which likely leads to immortalization of T cells. Tax is expressed and known to function in both the cell nucleus and the cytoplasm. Tax has defined nuclear localization (NLS) and nuclear export signals that enable shuttling between the two compartments. In this study, we identified a novel region in Tax that targets the protein to discrete nuclear foci that we have previously termed Tax speckled structures (TSS). We demonstrated that the identified region is both necessary and sufficient for directing proteins to TSS. This novel TSS localization signal (TSLS), spanning amino acids 50 to 75, is separable from and adjacent to the NLS of Tax. Coexpression of a Tax NLS mutant and a Tax TSLS mutant rescued the nuclear entry and subnuclear TSS targeting of both proteins, demonstrating that these signals are independent domains. Our analysis also revealed that Tax proteins deficient for dimerization fail to localize to the nucleus. Consequently, when we restored dimerization via induction of a heterologous "dimerizer" domain, nuclear localization was rescued. Thus, we defined additional domains in Tax specific for nuclear localization and subnuclear targeting. Our results reveal a more complex network for regulation of Tax subcellular localization and subsequent function.Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia as well as subneoplastic disease states, the most prevalent being HTLV-1-associated myelopathy/tropical spastic paraparesis (22,44,47,63,68). Tax is a potent transcriptional transactivator of both viral and cellular genes and can physically associate with a number of cellular proteins, thus altering their functions (10). Expression of Tax results in the dysregulation of critical cell functions, including cell cycle regulation, proliferation, DNA damage recognition and repair, and apoptosis. Furthermore, although the pathogenesis of adult T-cell leukemia is not completely understood, the transformation of T cells infected with HTLV-1 can be logically attributed to genomic instability resulting from the pleiotropic activities of the viral protein Tax (30, 41).Of particular note for this study, Tax has documented nuclear and cytoplasmic functions; thus, the regulation of Tax localization to subcellular compartments is a critical aspect of overall Tax function (11, 23, 52). Tax contains a nuclear localization signal (NLS) sequence in the N-terminal region of the protein that is necessary and sufficient for nuclear localization (23,53,55). We previously showed that Tax "shuttles" between nuclear and cytoplasmic subcellular compartments and identified a consensus nuclear export signal (NES) sequence defined by amino acids 190 to 203 (11). Subsequently, a fine-mapping mutational analysis of this region by Alefantis et al. (1) clearly demonstrated a functional NES at this site. These signals presumably mediate interactions between Tax and karyopherins in the nuclear pore comp...

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