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Originally published In Press as doi:10.1074/jbc.M512375200 on March 24, 2006

J. Biol. Chem., Vol. 281, Issue 20, 14041-14047, May 19, 2006
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Deregulated Activation of Oncoprotein Kinase Tpl2/Cot in HTLV-I-transformed T Cells*

Geetha Babu, Michael Waterfield, Mikyoung Chang, Xuefeng Wu, and Shao-Cong Sun1

From the Department of Microbiology and Immunology, Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Received for publication, November 17, 2005 , and in revised form, February 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-{kappa}B1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-{kappa}B by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adult T cell leukemia is a fatal T cell malignancy induced by infection of the human T cell leukemia virus type I (HTLV-I)2 (1, 2). The HTLV-I genome encodes a regulatory protein that has potent transforming activity and is required for HTLV-I-induced T cell transformation (3–5). Tax promotes cell transformation by inducing aberrant expression of many host genes involved in cell growth and survival (6, 7). Among the cellular genes activated by Tax are those coding for interleukin 2 (IL-2) and IL-2 receptor {alpha} subunit, which is likely required for the initial stage of T cell proliferation following HTLV-I infection. In addition, Tax also activates genes encoding other cytokines and cytokine receptors, cell cycle regulators, apoptosis inhibitors, and cell adhesion molecules. Tax induces cellular gene expression by deregulating the activity of transcription factors, most notably NF-{kappa}B (8).

NF-{kappa}B represents a family of inducible transcription factors that regulate diverse biological processes, including the growth and survival of both T cells and nonlymphoid cells (9–13). The NF-{kappa}B factors are normally sequestered in the cytoplasm by association with specific inhibitors belonging to the I{kappa}B family (14). Activation of NF-{kappa}B involves phosphorylation of I{kappa}Bs by a specific I{kappa}B kinase (IKK), which is composed of two catalytic subunits, IKK{alpha} and IKKbeta, and a regulatory subunit, IKK{gamma} (15). In response to various immune and stress stimuli, IKK is rapidly activated and mediates I{kappa}B phosphorylation and subsequent degradation, which allows NF-{kappa}B to move to the nucleus and exert its transcription function. Recent evidence suggests that NF-{kappa}B activation also involves phosphorylation of NF-{kappa}B members, especially the trans-activation subunit RelA. RelA phosphorylation is required for enhancing its transactivation function, thus allowing the {kappa}B enhancer-bound NF-{kappa}B to functionally mediate target gene transcription. Whereas NF-{kappa}B activation occurs transiently in a normal immune response, this cellular signaling pathway is persistently activated in freshly isolated adult T cell leukemia cells and T cell lines transformed by HTLV-I (6). We and others have previously shown that the Tax-mediated persistent activation of NF-{kappa}B involves its physical association with IKK via the IKK{gamma} subunit (16–18). Emerging evidence suggests that Tax also promotes the transactivation activity of NF-{kappa}B, although the underlying mechanism is not completely understood (19, 20).

Despite the extensive studies in Tax/host interaction, many missing links exist. For example, it is unclear whether Tax targets the activation of upstream kinases, especially those involved in the activation or modulation of NF-{kappa}B function. In the work presented here, we have shown that Tax induces the activation of a protooncoprotein kinase, Tpl2 (also known as Cot; hereafter referred to Tpl2/Cot). Tpl2/Cot is a member of the MAP kinase kinase kinase family initially identified as a transforming protein through functional cloning (21). The murine Tpl2/Cot was independently identified as a protooncogene activated by proviral insertion in T cell lymphomas induced by Moloney murine leukemia virus (22). The T cell-transforming activity of Tpl2/Cot appears to involve both its overexpression and its truncation at the carboxyl-terminal region (23). Transfection studies demonstrate that Tpl2/Cot activates a number of downstream signaling pathways, including those leading to activation of the ERK, c-Jun amino-terminal kinase (JNK), and p38 MAPK, IKK/NF-{kappa}B, and NF-AT (24–27). Although these findings provide important insight into the oncogenic potential of deregulated Tpl2/Cot, recent gene targeting studies reveal that Tpl2/Cot plays a physiological role in regulating the activation of MEK1 and its downstream target ERK in macrophages stimulated by bacterial lipopolysaccharides (LPS) and the inflammatory cytokine tumor necrosis factor {alpha} (28, 29).

We and others have recently shown that the signaling function of Tpl2/Cot is subject to tight regulation by the NF-{kappa}B1 precursor protein p105 (30, 31). In macrophages as well as various other cell types analyzed so far, Tpl2/Cot is physically associated with p105 (26, 30, 31). Through this molecular interaction, p105 both stabilizes Tpl2/Cot and inhibits its kinase function. Tpl2/Cot undergoes rapid degradation in p105-deficient cells derived from nf{kappa}b1 knock-out mice, which is associated with a defect in LPS-induced MEK/ERK signaling pathway. On the other hand, the p105-bound Tpl2/Cot is functionally inactive, and its activation involves signal-induced p105 degradation and the liberation of Tpl2/Cot. Strong evidence suggests that the signal-induced Tpl2/Cot activation requires IKKbeta, which functions by phosphorylating p105 and triggering the proteolysis of p105 (32, 33). Thus, pharmacological inhibitors of IKKbeta and proteasome are able to block LPS-stimulated activation of Tpl2/Cot and its downstream signaling pathways. Under normal conditions, the activated free Tpl2/Cot is rapidly degraded (30, 31), which appears to serve as a mechanism that prevents persistent activation of this oncoprotein kinase. Remarkably, we have found in the present study that Tpl2/Cot is constitutively activated in a panel of HTLV-I-transformed T cell lines. This deregulated cell signaling is at least partially mediated through the Tax protein, because expression of Tax in the absence of other viral proteins is sufficient to activate Tpl2/Cot. These results provide a novel insight into the molecular mechanism by which Tax deregulates cellular signaling pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Primary Cells—C8166 (34), HUT102 (35), MT-2 (36), and SLB-1 (37) are IL-2-independent HTLV-I-transformed T cell lines, and E55 is a HTLV-I-immortalized human T cell line that requires IL-2 for growth (38). These cell lines have been described previously (39). Human embryonic kidney 293 cells, human cervical carcinoma HeLa cells, and human leukemia T cell lines Jurkat, SupT1, and MOLT4 were also obtained from ATCC. Human peripheral blood mononuclear cells (PBMC) were prepared from Buffy coats as described (40). HTLV-I-immortalized PBMC was generated by in vitro co-cultivation of PBMC with {gamma}-irradiated MT-2 cells (41).

Plasmid Constructs, Antibodies, and Other Reagents—The pcDNA-HA-Tpl2/Cot was generated by PCR-based cloning of the human Tpl2/Cot cDNA into pcDNA-HA vector (17). The catalytically inactive Tpl2/Cot (Tpl2/Cot mut) was generated by site-directed mutagenesis to replace lysine 167 with methionine in pcDNA-HA-Tpl2/Cot. The {kappa}B-driven luciferase reporter ({kappa}B-TATA-luc) and pCMV4-based cDNA expression vectors encoding Tax, RelA, and p50 were provided by Dr. Warner Greene (42–44). The expression vector encoding Gal4-p65 fusion protein (pM-p65) was created by inserting the human p65 cDNA into the pM vector (Clontech). The Gal4 reporter plasmid (UAS-TK-Luc) was provided by Dr. Larry Jameson (45). The green fluorescence protein (GFP) expression vector (pEGFP) was from Clontech. To generate retroviral vectors encoding Tpl2/Cot and Tpl2/Cot mut, the corresponding cDNAs were subcloned into the pCLXSN retroviral vector (provided by Dr. Inder Verma, see Ref. 46). GST-MEK1 was described previously (32). The DNA constructs were partially sequenced at the Core Facility of Hershey Medical Center.

The carboxyl-terminal-specific anti-p105 antibody (anti-p105C) was provided by Dr. Nancy Rice. Horseradish peroxidase-conjugated HA antibody (3F10) was from Roche Applied Science. Anti-Tax monoclonal antibody was prepared from a hybridoma (168B17-46-34) provided by the AIDS Research and Reference Program, NIAID, National Institutes of Health. Phospho-specific MEK1 antibody (anti-MEK1-P) was from Cell Signaling. The antibody for Tpl2/Cot (anti-Cot M20), horseradish peroxidase-conjugated anti-Cot M20, and the other antibodies were from Santa Cruz Biotechnology. The IKKbeta inhibitor PS1145 was provided by Millennium Pharmaceuticals, Inc. (47). Phorbol 12-myristate 13-acetate, ionomycine, and LPS (derived from Escherichia coli 0127:B8) were from Sigma, and MG132 was from Alexis.

Cell Transfection and Retroviral Infection—Jurkat cells (1 x 106), HeLa cells (1 x 105, seeded in 6-well plates), and 293 cells (1 x 105, seeded in 6-well plates) were transfected using Lipofectamine-2000 (Invitrogen). Retroviral transduction was performed using the pCLXSN system provided by Dr. I. Verma (46). The procedure for retrovirus production and infection was as previously described (48) except for the inclusion of vesicular stomatitus virus glycoprotein (provided by Dr. T. Friedmann) (49) in the packaging. The infected cells were enriched by drug selection using neomycin.

Immunoblotting (IB) and in Vitro Kinase Assays—Cells were lysed in a kinase cell lysis buffer containing 20 mM Hepes (pH 7.6), 250 mM NaCl, 0.5% Nonidet P-40, 20 mM beta-glycerophosphate, 1 mM EDTA, 20 mM p-nitrophenylphosphate, 0.1 mM Na3VO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Sigma). The Tpl2/Cot complexes were isolated by immunoprecipitation (IP) and subjected to in vitro kinase assays essentially as described (50). Briefly, the IP beads were washed twice with kinase cell lysis buffer and twice with kinase reaction buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM beta-glycerophosphate, 1 mM EDTA, and 2 mM dithiothreitol). Kinase assays were initiated by addition to the beads of 30 µl of kinase reaction buffer containing 20 µM ATP, 2.5 µCi [{gamma}-32P]ATP, and 1 µg of substrate (GST-MEK1). After 30 min of incubation at 30 °C, the phosphorylated substrates and autophosphorylated Tpl2/Cot were fractionated by SDS-PAGE and visualized by autoradiography.

For p105 depletion, the cell lysates were incubated with anti-p105C antibody and protein A-agarose for 1 h followed by removing the p105 immune complexes by centrifugation. This procedure was repeated two more times to achieve the maximal p105 depletion. The p105-depleted cell lysates were then subjected to Tpl2/Cot kinase assays as described above.

For IB assays, cell lysates were subjected to SDS-PAGE, and the proteins were transferred onto nitrocellulose and detected by specific antibodies using the enhanced chemiluminescent system. For detecting MEK1 phosphorylation, the cells were starved overnight in medium containing low serum (0.5%) before preparation of the cell lysates.

Reporter Gene Assays—Approximatedly 1.25 x 106 Jurkat cells were transfected with the indicated cDNA expression vectors together with 25 ng of {kappa}B-luciferase reporter. For normalizing the transfection efficiency, the cells were also transfected with a control Renilla luciferase reporter driven by the constitutive thymidine kinase promoter (5 ng). At 40 h post-transfection, the cells were collected for duel luciferase assays (Promega). The {kappa}B-specific luciferase activity was normalized based on the Renilla luciferase activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive Tpl2/Cot Activation in HTLV-I-transformed T Cells—We examined the activity of Tpl2/Cot in HTLV-I-transformed T cells by immune complex kinase assays. As a positive control, we used LPS-stimulated RAW264 macrophage cell line. As expected, the kinase activity of Tpl2/Cot was hardly detectable in RAW 264 cells under unstimulated conditions but could be stimulated by LPS (Fig. 1A, top panel, lanes 1 and 2). Interestingly, however, constitutive Tpl2/Cot kinase activity was detected in a panel of HTLV-I-transformed T cell lines (lanes 6–10), including both IL-2-independent (C8166, MT2, SLB-1, HUT102) and IL-2-dependent (E55) lines. In contrast, no significant Tpl2/Cot activity was detected in several HTLV-I-negative T cell lines, including Jurkat, SupT1, and MALT4 (lanes 3–5). Parallel IB was performed to measure the expression of Tpl2/Cot proteins. As previously reported (21, 51), Tpl2/Cot was expressed as two isoforms, Tpl2/CotL and Tpl2/CotS, denoting longer and shorter isoforms, respectively. The steady level of Tpl2/Cot in these cells was similar to that of HTLV-transformed T cells, although the HTLV-negative cell lines have very low levels of the short isoform of Tpl2/Cot (Tpl2/CotS) (Fig. 1A, middle panel). The low level of Tpl2/CotS expression did not seem to contribute to the lack of Tpl2/Cot kinase activity in the HTLV-I-negative cells, because normal human T cells from fresh PBMCs expressed both the long (Tpl2/CotL) and short (Tpl2/CotS) isoforms of Tpl2/Cot and did not exhibit significant constitutive Tpl2/Cot activity (Fig. 1B, upper panel, lane 1).


Figure 1
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  		FIGURE 1.
Constitutive activation of Tpl2/Cot in HTLV-I-transformed T cells. A, Tpl2/Cot was isolated from the indicated HTLV-I-positive and -negative T cell lines followed by in vitro kinase assays using GST-MEK1 as template (top panel). The Tpl2/Cot proteins isolated from non-treated (NT) and LPS-stimulated (15 min) Raw264 macrophage cell line were used as negative and positive controls, respectively. The expression level of the two isoforms of Tpl2/Cot was detected by IB (middle panel), and a tubulin IB was used as a loading control (bottom panel). B, Tpl2/Cot was isolated from human PBMC, PBMC stimulated for 30 min with phorbol 12-myristate 13-acetate plus ionomycin, PBMC immortalized by HTLV-I, or the HTLV-I-transformed C8166 cells followed by in vitro kinase assays (upper panel). The Tpl2/Cot expression was detected by IB (lower panel).

 


Figure 2
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  		FIGURE 2.
The active pool of Tpl2/Cot in HTLV-I-transformed T cells is not complexed with p105. A, a large proportion of Tpl2/Cot is not associated with p105 in HTLV-I-transformed T cells. Cell lysates derived from normal human PBMC or the HTLV-I-transformed C8166 cells were subjected to IP with anti-p105C followed by IB to detect the expression levels of p105 (top panel) and p105-associated Tpl2/Cot (middle panel). The total Tpl2/Cot protein was analyzed by IP with anti-Cot followed by IB with the same antibody (bottom panel). B, depletion of p105 and its associated Tpl2/Cot. C8166 cell lysates were precleared by three rounds of IP using either a preimmune serum or anti-p105C. The precleared cell lysates were then subjected to IP using anti-p105C followed by IB to detect the precipitated p105 (upper panel) and its associated Tpl2/Cot (lower panel). C, depletion of p105 had no significant effect on Tpl2/Cot activity. The preimmune and p105-depleted C8166 cell lysates used in panel B were subjected to IP using either anti-Cot or anti-p105C followed by in vitro kinase assays to determine the catalytic activity of Tpl2/Cot (upper panel) and IB to detect the free and p105-associated Tpl2/Cot (lower panel). The phosphorylated substrate (GST-MEK1-P) and autophosphorylated kinases (Tpl2/CotL-P and Tpl2/CotS-P) are indicated.

 
Tpl2/Cot activation has been extensively studied in macrophages, where its activity is potently stimulated by LPS. It remains unclear whether Tpl2/Cot is activated in T cells by the T cell receptor signals. We examined this possibility by stimulating human PBMC with phorbol 12-myristate 13-acetate and ionomycin, pharmacological agents that stimulate the protein kinase C and calcium signals downstream of T cell receptors. Upon stimulation with phorbol 12-myristate 13-acetate and ionomycin, we consistently detected a weak but significant increase in Tpl2/Cot kinase activity (Fig. 1B, upper panel, lane 2 and data not shown). This finding suggests the possible involvement of Tpl2/Cot in normal T cell activation. However, parallel kinase assays using the same amounts of cell lysate of HTLV-I-transformed C8166 cell line revealed a much more robust activation of Tpl2/Cot (lane 3). Significant Tpl2/Cot activity was also readily detected in PBMC immortalized with HTLV-I (lane 4). The levels of Tpl2/Cot in the normal and HTLV-I-transformed T cells were comparable (Fig. 1B, lower panel). Thus, the oncoprotein kinase Tpl2/Cot is potently activated in HTLV-I-transformed T cells.

Active Tpl2/Cot Is Not Bound by p105—In primary bone marrow-derived macrophages and macrophage cell lines, Tpl2/Cot is stabilized through its physical association with p105 (26, 30, 31). Within this stable complex, the MEK kinase activity of Tpl2/Cot is inhibited by p105 (30, 31). To understand the mechanism of constitutive Tpl2/Cot activation by HTLV-I, we examined the association of Tpl2/Cot with p105 in the HTLV-I-transformed T cell line C8166. IP was performed with either anti-p105C or anti-Tpl2/Cot antibody, followed by IB detection of the p105-associated Tpl2/Cot and total Tpl2/Cot, respectively. As expected from the result presented in Fig. 1B, comparable amounts of total Tpl2/Cot were detected from PBMC and HTLV-I-transformed C8166 cells (Fig. 2A, bottom panel). Importantly, however, the p105-associated Tpl2/Cot was significantly less in C8166 cells than in the PBMC (middle panel). The level of p105 was also lower in C8166 cells (top panel) as well as in the other HTLV-I-transformed T cells (data not shown). This finding suggested that a large proportion of Tpl2/Cot was not associated with p105 in the HTLV-I-transformed T cells. To confirm this idea, we removed the p105 complexes from C8166 cell lysates by antibody depletion assays. As shown in Fig. 2B, p105 was efficiently depleted after three rounds of IP using the anti-p105C antibody (upper panel, lane 2). As expected, the p105-associated Tpl2/Cot proteins were also depleted (lower panel, lane 2). However, parallel kinase assays revealed that the p105 depletion did not reduce the kinase activity of Tpl2/Cot (Fig. 2C, upper panel, lane 3), thus supporting the idea that the active form of Tpl2/Cot is free from p105 (30–33). Consistently, after p105 depletion substantial amounts of Tpl2/Cot were still precipitated by the anti-Tpl2/Cot antibody (lower panel, lane 3) but not by the anti-p105C antibody (lane 4). Together, these data suggest that activation of Tpl2/Cot in HTLV-infected T cells involves the liberation of Tpl2/Cot from its inhibitor p105.


Figure 3
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  		FIGURE 3.
The active Tpl2/Cot in HTLV-I-transformed T cells is stable and insensitive to inhibitors of proteasome and IKKbeta. A, Tpl2/CotS is stable and largely contributes to the constitutive Tpl2/Cot activity in HTLV-I-transformed T cells. C8166 and MT2 cells were incubated for the indicated times with a protein synthesis inhibitor, cycloheximide (5 µg/ml). Tpl2/Cot was isolated from the cell lysates by IP using anti-Cot followed by in vitro kinase assays using GST-MEK1 as substrate (top panel). The phosphorylated substrate and autophosphorylated Tpl2/Cot isoforms are indicated. The expression levels of Tpl2/Cot isoforms as well as a control protein, I{kappa}B{alpha}, were detected by IB (middle and bottom panels). Tpl2/CotL, but not Tpl2/CotS, underwent rapid degradation. B, C8166 cells were incubated for the indicated times with either the proteasome inhibitor MG132 (25 µM) or the IKKbeta inhibitor PS1145 (10 µM) followed by Tpl2/Cot in vitro kinase assays (top panel) as described in panel A. The expression levels of Tpl2/Cot, p105, and I{kappa}B{alpha} were measured by IB (lower panels).

 
Active Tpl2/CotS Is Stable in HTLV-I-infected T Cells—Under normal conditions, the stability of Tpl2/Cot is dependent on its association with p105 (30, 31). In response to LPS stimulation, the longer Tpl2/Cot isoform is rapidly dissociated from p105, which correlates with catalytic activation of Tpl2/Cot. However, the active form of Tpl2/Cot is degraded within a short time, which may serve to prevent persistent activation of this oncogenic kinase. Because Tpl2/Cot is constitutively active and dissociated from p105 in HTLV-I-transformed T cells, it indicated that the pathologically activated Tpl2/Cot is either stabilized or undergoing rapid resynthesis in these tumor cells. To examine these possibilities, we incubated the cells with a protein synthesis inhibitor, cycloheximide, followed by kinase assays and IB to examine the kinase activity and expression of Tpl2/Cot proteins. We performed these studies using two HTLV-I-transformed T cell lines, C8166 and MT2. Incubation of these cells with cycloheximide resulted in rapid degradation of Tpl2/CotL (Fig. 3A, middle panel), which was reminiscent of its degradation in LPS-stimulated macrophages (30). This finding suggests that Tpl2/CotL is undergoing rapid degradation and resynthesis. Interestingly, however, the level of Tpl2/CotS was not significantly altered following cycloheximide treatment (Fig. 3A, middle panel) despite the dissociation of a large proportion of this Tpl2/Cot isoform from p105 (see Fig. 2A and 2C, lower panel). Consistently, the kinase activity of Tpl2/Cot was not appreciably affected by the cycloheximide treatment (Fig. 3A, top panel). This result was not due to inefficient inhibition of protein synthesis, because the cycloheximide treatment led to significant loss of I{kappa}B{alpha}, an NF-{kappa}B inhibitor known to be rapidly degraded in HTLV-I-transformed T cells (53–55). Thus, the short isoform of Tpl2/Cot is stable and contributes largely to the constitutive kinase activity of Tpl2/Cot in HTLV-I-transformed T cells.

Active Tpl2/Cot Is Insensitive to Inhibitors of IKKbeta and Proteasome—The activation of Tpl2/Cot in macrophages involves IKKbeta-mediated phosphorylation and degradation of p105 (32, 33). IKKbeta has also been implicated in Tpl2/Cot activation through other mechanisms (56). Of note, the HTLV-I-infected T cells have constitutively activated IKK (39, 57–59) and lower steady levels of p105 compared with uninfected cells (Fig. 2A, top panel). Thus, we suspected that IKK activation and p105 degradation might contribute to the persistent activation of Tpl2/Cot in HTLV-I-infected T cells. To explore this potential mechanism, we examined whether inhibitors of IKKbeta and proteasome affect the activation of Tpl2/Cot in these malignant T cells. Surprisingly, incubation of C8166 cells with the IKKbeta inhibitor PS1145 or the proteasome inhibitor MG132 for up to 4 h did not affect Tpl2/Cot kinase activity (Fig. 3B, top panel). Nor did these treatments result in the accumulation of p105 protein (Fig. 3B, third panel), suggesting that in HTLV-I-infected cells, p105 protein synthesis and rate of turnover are inherently lower compared with uninfected cells. It is therefore likely that the constitutive Tpl2/Cot activity seen in these cells is not due to rapid turnover of p105. Although PS1145 and MG132 treatments did not affect p105 protein levels, they resulted in accumulation of the labile I{kappa}B{alpha} molecule (Fig. 3B, bottom panel), confirming the effectiveness of these drugs. Interestingly, the level of Tpl2/CotL was also elevated in cells treated with both MG132 and PS1145 (Fig. 3B, second panel), suggesting the involvement of IKKbeta and proteasome in mediating Tpl2/CotL degradation in HTLV-I-infected T cells. Together, these results suggest that the constitutive activation of Tpl2/Cot is insensitive to inhibitors of IKKbeta and proteasome, at least during the incubation times (up to 4 h) used. We found that longer incubation times were toxic to the cells. However, it is still possible that the initial phase of Tpl2/Cot activation in HTLV-I-infected T cells may involve IKKbeta-mediated p105 phosphorylation and degradation, as suggested by the following studies.

HTLV-I-encoded Tax Protein Stimulates Tpl2/Cot Activation—HTLV-I-encoded Tax protein is largely responsible for HTLV-I-mediated deregulation of cellular gene expression. We thus examined whether Tax was able to stimulate the activity of Tpl2/Cot. For these studies, we used HeLa cells because of their high transfection efficiency as well as their expression of both isoforms of Tpl2/Cot. The cells were transfected with either control GFP or Tax, followed by examining Tpl2/Cot activation by kinase assays. Expression of Tax, but not GFP, resulted in significant activation of Tpl2/Cot, as demonstrated by its enhanced activity in substrate phosphorylation (Fig. 4A, top panel, MEK1-P) and autophosphorylation (CotL-P and CotS-P). This result was not due to enhanced expression of Tpl2/Cot, because the Tpl2/Cot protein level was even moderately reduced in the Tax-transfected cells (Fig. 4A, second panel). Thus, Tax is sufficient for inducing the activity of Tpl2/Cot.

To investigate the mechanism by which Tax stimulates Tpl2/Cot activation, we examined whether Tax induced p105 degradation and dissociation of the Tpl2/Cot-p105 complex. When coexpressed in HeLa cells, Tax stimulated the loss of wild-type p105 (Fig. 4B, top panel, lane 2) and the release of Tpl2/Cot (middle panel, lane 2). This effect was specific, because Tax did not alter the level of a phosphorylation-deficient p105 mutant (p105 S/A) known to be resistant to LPS-stimulated degradation (32). Consistently, Tax did not induce the release of Tpl2/Cot from p105 S/A. Further, the Tax-stimulated release of Tpl2/Cot from wild-type p105 was associated with a reduction in the level of Tpl2/Cot protein (Fig. 4B, bottom panel), suggesting degradation of Tpl2/Cot coupled with its dissociation from p105. These results indicate that Tax-stimulated Tpl2/Cot activation may involve IKKbeta-mediated phosphorylation of p105 and p105 degradation. This finding also suggests the possibility that the initial phase of Tpl2/Cot activation in HTLV-I-infected T cells may involve IKKbeta-mediated p105 phosphorylation and degradation.


Figure 4
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  		FIGURE 4.
Activation of Tpl2/Cot and MEK1 by Tax. A, HeLa cells were transfected with expression vectors encoding either GFP or Tax. Tpl2/Cot was isolated by IP with anti-Cot and subjected to in vitro kinase assays using GST-MEK1 (top panel). The phosphorylated substrate and autophosphorylated Tpl2/Cot isoforms are indicated. Parallel IB was performed to monitor the expression of Tpl2/Cot, Tax, and the loading control tubulin. B, Tax induces the dissociation of Tpl2/Cot from p105. HeLa cells were transfected with the expression vectors indicated below the figure (V, empty vector). The p105-associated Tpl2/Cot was isolated by IP using anti-p105C followed by IB detection of the precipitated p105 and the coprecipitated Tpl2/Cot (by horseradish peroxidase-conjugated anti-HA). The level of Tpl2/Cot in the lysates was analyzed by direct IB (bottom panel). Note that the Tpl2/Cot expression vector (labeled Cot) encodes HA-tagged full-length human Cot, which is equivalent to Tpl2/CotL. Tpl2/CotS was not detected with the anti-HA antibody due to its lack of amino-terminal sequence (data not shown). C, MEK1 phosphorylation in HTLV-I-transformed T cells. Cell lysates from HTLV-positive (+) and HTLV-negative (–) T cell lines were subjected to IB using either phospho-specific (upper panel) or pan (lower panel) anti-MEK1 antibodies. D, induction of MEK1 phosphorylation by Tax. HeLa cells were transfected with cDNA expression vectors encoding either GFP or Tax. Cell lysates were subjected to IB using the phospho-specific ({alpha}MEK1-P) or regular ({alpha}MEK1) MEK1 antibodies or the anti-Tax antibody.

 
Tax Stimulates MEK1 Phosphorylation—As noted in the Introduction, MEK1 is the predominant substrate of Tpl2/Cot under physiological conditions, although Tpl2/Cot also phosphorylates additional downstream kinases under overexpression conditions. Because of the potent activation of Tpl2/Cot in HTLV-I-infected T cells, we examined whether MEK1 is activated in these infected cells. Upon serum starvation, the control Jurkat and MOLT4 cells did not express appreciable levels of phospho-MEK1 (activated form) (Fig. 4C, upper panel, lanes 1 and 2). On the other hand, the HTLV-I-infected cells expressed different levels of phospho-MEK1 (lanes 3–6), thus supporting the role of Tpl2/Cot in MEK1 activation. However, it should be noted that the level of MEK1 phosphorylation did not tightly correlate with that of Tpl2/Cot activation (Fig. 1, compare panels A and C), which could be due to the differential activity of other MEK1 regulators, such as MAP kinase kinase kinase and negative regulatory phosphatases.

We next examined whether Tax was able to stimulate MEK1 phosphorylation. Expression of the control GFP did not induce MEK1 phosphorylation, whereas expression of Tax led to significant MEK1 phosphorylation (Fig. 4D). Thus, the activation of Tpl2/Cot by Tax and HTLV-I is correlated with in vivo phosphorylation of MEK1, a mechanism that mediates activation of this MAPK kinase. Future studies will determine whether Tpl2/Cot is required for Tax-stimulated MEK1 phosphorylation.

Tpl2/Cot Synergizes with Tax in the Stimulation of NF-{kappa}B Transactivation Activity—One important cellular target of Tax is the NF-{kappa}B transcription factor. We thus examined whether Tpl2/Cot participates in Tax-mediated NF-{kappa}B activation by performing {kappa}B-specific reporter gene assays. As expected, expression of Tax in Jurkat T cells resulted in stimulation of {kappa}B reporter activity in a dose-dependent manner (Fig. 5B, columns 1–4). Interestingly, expression of wild-type Tpl2/Cot potently enhanced the {kappa}B activation by Tax (columns 5–8), whereas expression of a catalytically inactive Tpl2/Cot mutant inhibited the Tax-induced {kappa}B activation (columns 9–12). These results suggested that Tpl2/Cot synergizes with Tax in the activation of NF-{kappa}B. Parallel reporter gene assays using individual NF-{kappa}B subunits revealed that Tpl2/Cot potently enhanced the {kappa}B reporter gene activation by RelA (Fig. 5C, column 6), a central component of the NF-{kappa}B complex. Tpl2/Cot alone only weakly induced the {kappa}B reporter activity (column 2), and Tpl2/Cot did not synergize with the p50 subunit of NF-{kappa}B(column 4).

To further investigate the mechanism by which Tpl2/Cot participates in Tax-stimulated NF-{kappa}B activation, we examined whether Tpl2/Cot regulates the transactivation activity of RelA. These studies were performed using a fusion gene construct encoding the DNA-binding domain of yeast Gal4 and human RelA (Gal4-RelA). Transactivation of a Gal4-specific luciferase reporter is mediated by the Gal4 DNA binding and the RelA transactivation functions. When expressed in Jurkat cells, Tpl2/Cot weakly stimulated the transactivation activity of RelA (Fig. 5D, columns 2–4). Stronger activation was detected with Tax (column 5). Remarkably, however, Tpl2/Cot potently synergized with Tax in the induction of RelA transactivation activity (columns 6–8). Together, these results suggest that Tpl2/Cot participates in Tax-stimulated activation of RelA transactivation activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Tpl2/Cot was originally identified as a protooncogene activated by proviral insertion in Moloney murine leukemia virus-transformed rodent T cell lymphomas (22) and a potent transforming gene when expressed in cell line or transgenic mice (21–23). Recent studies revealed that the Tpl2/Cot gene expression (mRNA level) is enhanced in breast cancer cells and large granular leukemia cells (60, 61). The results presented in this report suggest the possibility that deregulation of Tpl2/Cot in cancer cells may also occur posttranslationally. We have shown that Tpl2/Cot is constitutively activated in human T cell lines transformed by the oncogenic retrovirus HTLV-I. In contrast to the cis-acting transformation mechanism of Moloney murine leukemia virus, HTLV-I transforms human T cells by a transactivating mechanism involving the oncogenic action of its Tax proteins (62). Tax acts by activating host transcription factors, most notably NF-{kappa}B, leading to deregulated expression of various genes involved in the control of cell growth and survival (8). The constitutive activation of Cot/Tp2 in HTLV-I-transformed T cells appears to also involve Tax, because Tax stimulates the kinase activity of Tpl2/Cot as well as in vivo phosphorylation of its substrate, MEK1, in transfected cells. Further, we have obtained evidence that Tpl2/Cot participates in Tax-stimulated activation of NF-{kappa}B.

The mechanism of Tpl2/Cot activation has been mainly studied using murine macrophages stimulated with the bacterial component LPS (30, 31). Using this model system, we and others have recently shown that the kinase activity of Tpl2/Cot is tightly regulated through its physical interaction with the NF-{kappa}B1 precursor protein, p105. P105 functions to both stabilize Tpl2/Cot and inhibit its kinase activity. Thus, activation of Tpl2/Cot under normal conditions involves its release from p105 as a result of IKKbeta-mediated degradation of p105 (32, 33). It is remarkable that a large proportion of Tpl2/Cot is free from p105 in HTLV-I-transformed T cells and contributes to the constitutive Tpl2/Cot kinase activity in these cancer cells. This pathological mechanism of Tpl2/Cot activation is likely due to the low steady level of p105 in the HTLV-I-transformed T cells. Because IKK is constitutively activated in these leukemia cells (8), we speculated that the low level of p105 might result from IKK-mediated degradation. Surprisingly, however, we did not detect significant restoration of p105 in HTLV-I-transformed T cells after incubation with inhibitors of IKKbeta or proteasome for up to 4 h. Because of the toxicity of these drugs, we were unable to perform longer times of incubation. Thus, it remains possible that IKKbeta mediates slow and persistent degradation of p105 in the HTLV-I-transformed cell lines. On the other hand, it is also likely that the low level of p105 in HTLV-I-transformed T cells is because of suppression of nfkb1 gene expression.


Figure 5
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  		FIGURE 5.
Tpl2/Cot participates in Tax-stimulated NF-{kappa}B activation. A, expression of Tpl2/Cot in Jurkat T cells via retroviral transduction. Jurkat cells were infected with either an empty pCLXSN retroviral vector or the same vector encoding HA-tagged wild-type Tpl2/Cot or a catalytically inactive Tpl2/Cot mutant. IB was performed using anti-HA to detect the exogenous Tpl2/Cot proteins. B, Tpl2/Cot participates in Tax-stimulated activation of NF-{kappa}B transcription activity. The retrovirus-infected Jurkat T cells shown in panel A were transfected with increasing doses of Tax expression vector along with a {kappa}B luciferase reporter (25 ng) and a control Renilla luciferase reporter driven by the constitutive thymidine kinase promoter (5 ng). The {kappa}B-specific luciferase activity was normalized based on the control Renilla luciferase activity and presented as -fold induction. C, Tpl2/Cot synergizes with RelA. Jurkat T cells were transfected with either an empty vector or the indicated cDNA expression vectors (100 ng) along with {kappa}B luciferase and the control Renilla luciferase reporters. The {kappa}B-specific luciferase activity was determined as described in panel B. D, Tpl2/Cot synergizes with Tax to induce RelA transactivation activity. Jurkat cells were transfected with increasing amounts of Tpl2/Cot expression vector either in the absence (–) or presence (+) of a Tax expression vector. All the cells were also transfected with an expression vector encoding Gal4-p65 (100 ng), a Gal4 luciferase reporter (UAS-TK-Luc, 100 ng), and the control Renilla luciferase reporter (20 ng). Data shown in panels B–D are mean ± S.E. from three to four independent experiments.

 
Under normal conditions, Tpl2/Cot is rapidly degraded unless it is bound by p105 (30, 31). Interestingly, a large proportion of Tpl2/CotL and Tpl2/CotS is dissociated from p105 in HTLV-I-transformed cells. However, Tpl2/CotS is highly stable, whereas Tpl2/CotL undergoes rapid degradation and resynthesis. This finding indicates that Tpl2/Cot may be stabilized by a p105-independent mechanism in HTLV-I-transformed cells. The degradation of Tpl2/CotL in HTLV-I-transformed cells is unlikely mediated solely through its release from p105, because this proteolytic process is dependent on IKKbeta. Although how IKKbeta selectively mediates degradation of the long isoform of Tpl2/Cot is not understood, Tpl2/CotL is known to contain an extra amino-terminal region composed of 30 amino acids. Thus, one intriguing possibility is that the amino-terminal unique region of Tpl2/CotL may serve as a site of interaction or phosphorylation for IKKbeta or an IKKbeta-dependent kinase.

Tpl2/Cot has been shown to target the activation of a variety of downstream signaling pathways, including those leading to activation of MAPKs, NF-{kappa}B, and NF-AT. Of note, most of these pathways are activated in HTLV-I-transformed T cells (8, 52, 63). In the present study, we have shown that the MAPK kinase MEK1 is also constitutively activated in the HTLV-I-transformed T cells and the activation of MEK1 is stimulated by Tax. Because Tpl2/Cot is a known regulator of MEK1, this finding implicates a role for Tpl2/Cot in mediating Tax-stimulated MEK1 activation. Of course, it remains to be examined whether Tpl2/Cot is indeed required for MEK1 activation in HTLV-I-transformed or Tax-expressing cells. Nevertheless, our data strongly suggest that Tpl2/Cot participates in Tax-stimulated activation of NF-{kappa}B. In transfected cells, Tpl2/Cot synergizes with Tax in the activation of NF-{kappa}B reporter gene. Consistent with prior studies (27), Tpl2/Cot alone was able to induce a low level of NF-{kappa}B activity. However, when expressed together with Tax, this oncoprotein kinase induces robust NF-{kappa}B transcription activity. We have obtained evidence that Tpl2/Cot regulates the transactivation activity of RelA and that this function of Tpl2/Cot can be potently enhanced by Tax.

Based on the findings discussed above, we propose the following working model. In HTLV-I-transformed cells, p105 steady level is reduced through p105 degradation and/or nf{kappa}b1 gene suppression. The low level of p105 expression results in generation of constitutively active form of Tpl2/Cot, which appears to be stabilized through a p105-independent mechanism. Active Tpl2/Cot participates in Tax-stimulated activation of NF-{kappa}B by enhancing the transactivation activity of RelA. The activation of Tpl2/Cot in HTLV-I-transformed cells likely involves the action of the Tax protein.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 CA68471 and R01 AI057555 (to S.-C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 717-531-4164; Fax: 717-531-6522; E-mail: sxs70{at}psu.edu.

2 The abbreviations used are: HTLV-I, human T cell leukemia virus type I; IL-2, interleukin-2; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; GFP, green fluorescence protein; GST, glutathione S-transferase; IP, immunoprecipitation; IB, immunoblotting; ERK, extracellular signal-regulated kinase; HA, hemagglutinin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Warner Greene for Tax and NF-{kappa}B expression vectors, Dr. Nancy Rice for anti-p105C antibody, Dr. Inder Verma for retroviral vectors, Dr. Larry Jameson for UAS-TK-Luc, Millennium Pharmaceuticals, Inc. for the IKKbeta inhibitor PS1145, and the AIDS Research and Reference Program of NIAID, National Institutes of Health for anti-Tax hybridoma.



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