Site-specific Phosphorylation Differentiates Active from Inactive Forms of the Human T-cell Leukemia Virus Type 1 Tax Oncoprotein

Abstract

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...

Peptidylproline cis - trans -Isomerase Pin1 Interacts with Human T-Cell Leukemia Virus Type 1 Tax and Modulates Its Activation of NF-κB

“…Pin1 recognizes phosphorylated proteins. Tax is a phosphoprotein, which is phosphorylated on serine residue(s) in both mouse and human cells (12,13,27). We wondered next whether Pin1 might interact with Tax.…”

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

“…10,11 Posttranslational modification of proteins regulates protein functions by modifying their subcellular localization, stability, and/or network of interaction. We and others have described different forms of Tax posttranslational modifications including phosphorylation, 12,13 acetylation, 14 ubiquitylation, and small ubiquitin-like modifier (SUMO)ylation, 15,16 all of which are implicated in Tax-mediated activation of gene expression. Indeed, we showed that Tax is differentially ubiquitylated by either K-48 ubiquitin chains leading to Tax degradation by the proteasome or by K-63 ubiquitin chains that mediates IKK recruitment to the centrosome and IKK activation.…”

Human T-cell Leukemia Virus Type 1 Tax Oncoprotein Prevents DNA Damage-induced Chromatin Egress of Hyperphosphorylated Chk2

“…This interaction was determined via immunoprecipitation following cotransfection of plasmids expressing both Tax and Chk2 and by co-immunoprecipitation following immunoprecipitation of endogenous Chk2. In the studies described herein, we used affinity purification of S-Tax using a system we recently described (35). In this study, the affinity isolation of Tax protein from cells resulted in a multiprotein complex containing endogenous Chk2 as a component (Fig.…”