Tyrosine Phosphorylation of the Tio Oncoprotein Is
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病菌学杂志 2005年第16期
Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universitt Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany
ABSTRACT
Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.
INTRODUCTION
Herpesvirus ateles (ateline herpesvirus 3), a monkey herpesvirus that is apathogenic in its natural host, the spider monkey (Ateles sp.), induces fatal T-cell lymphomas in other New World monkey species (16, 26, 37). Continuous T-cell lines have been established from infected animals. Infection of peripheral blood lymphocyte cultures resulted in permanently growing populations of T cells from various monkey species (18). These transforming properties are reminiscent of herpesvirus saimiri (saimiriine herpesvirus 2), the prototype of the Rhadinovirus family.
Nucleotide sequence variability in a distinct genomic locus of herpesvirus saimiri led to the classification of herpesvirus saimiri isolates into subgroups A, B, and C (12, 35, 36). While all subgroups are oncogenic in highly susceptible monkeys, other animal species and in vitro transformation experiments revealed differences in the oncogenic potential (5, 35, 46). Herpesvirus saimiri subgroup C strains, but neither subgroup A or B strains nor herpesvirus ateles isolates, are capable of transforming human T cells to permanent growth in cell culture (2, 5).
Targeted and spontaneous deletions within the herpesvirus saimiri genome indicated that proteins encoded within the subgroup-defining hypervariable region are not required for virus replication but are essential for the oncogenic phenotype of these viruses in culture and in vivo (11, 13, 14, 40). Subgroup A and B genomes encode a single saimiri transformation-associated protein, StpA and StpB, respectively (9, 25, 33, 40). In subgroup C genomes, two open reading frames were identified, encoding StpC and the tyrosine kinase-interacting protein (Tip) (6, 14).
Sequence analysis revealed the colinearity of the genomes of herpesvirus ateles and herpesvirus saimiri. A spliced gene termed tio was identified within the hypervariable region of herpesvirus ateles. The amino acid sequence encoded by this gene displayed moderate similarities to both herpesvirus saimiri subgroup C oncoproteins StpC and Tip (1, 3). Recently, we demonstrated that Tio indeed functionally replaces StpC and Tip in the process of human and monkey T-cell transformation in vitro. These data strongly suggest Tio is the oncoprotein mediating the transforming phenotype of herpesvirus ateles (2).
StpC is a 102-amino-acid protein composed of a charged N terminus followed by 18 consecutive collagen repeats and a hydrophobic C-terminal membrane anchoring sequence. The first 17 amino acids display a net negative charge and a consensus motif for tumor necrosis factor-associated factor (TRAF) binding. StpC was shown to associate with cellular Ras competing for Raf-1 binding and to activate the NF-B signaling cascade (27, 32, 45). StpC appears to be an oncoprotein, as demonstrated by the transformation of rodent fibroblast cells in culture (29) and by the development of epithelial tumors in StpC transgenic mice (31, 39), but T-cell-specific functions have not been identified so far.
Tip, the tyrosine kinase-interacting protein, interacts with the nonreceptor tyrosine kinase Lck and thereby induces various downstream signals. Its functions contributing to the control of T-cell transformation may include modulation of the T-cell receptor and its downstream signals, gamma interferon induction, and STAT factor phosphorylation (7, 8, 38, 41, 42, 47). Tip expression alone was found to be lethal in transgenic mice. However, when a conditional expression cassette was used, the mice developed fulminant T-cell lymphomas, demonstrating the T-cell specificity of Tip (48).
Tio is a 269-amino-acid protein with a negatively charged amino terminus followed by a glycine-proline-rich sequence, including some interspersed collagen-like triplets and a potential TRAF-binding motif within the amino-terminal third. This indicates a structural similarity to StpC, however, a functional correlation has not been identified. A sequence highly homologous to the Tip protein was found in the carboxy-terminal third of Tio, which corresponds to the SH3-binding motif of Tip. Coimmunoprecipitation analysis revealed that Tio interacts with nonreceptor protein tyrosine kinases of the Src family (3), suggesting that Tio may exhibit Tip-related functions in T cells.
In this study, we analyzed the relevance of the Tio-Src kinase interaction for induction and maintenance of T-cell transformation by Tio-expressing herpesvirus saimiri recombinants. We demonstrate that the Src kinase activity and Tio interaction as well as Tio phosphorylation on a single tyrosine residue are essential for the T-cell-transforming potential of Tio.
MATERIALS AND METHODS
Generation of Tio mutants and recombinant viruses. The pcDNA3-derived plasmid coding for Flag-tagged Tio (3) was modified according to the Quick Change protocol (Stratagene, Amsterdam, The Netherlands) using primer pairs 5'-CCTCCACCACAATTAGCACCTGGGCCAGCAAACC-3' and 5'-GGTTTGCTGGCCCAGGTGCTAATTGTGGTGGAGG-3' to replace P292 with A and R294 with G, 5'-CTAAAGGCGGTGGGTAATCTTCATTTTTAGAATTAATG-3' and 5'-CATTAATTCTAAAAATGAAGATTACCCACCGCCTTTAG-3' for Y49F, 5'-GCATCACGACAATATTCATCCCTTGGGAGGACGC-3' and 5'-GCGTCCTCCCAAGGGATGAATATTGTCGTGATGC-3' for Y136F, 5'-CCCTTTTAATAAATTCCCAAAAAACTATAAAAAACTTAG-3' and 5'-CTAAGTTTTTTATAGTTTTTTGGGAATTTATTAAAAGGG-3' for Y167F, and 5'-CCCAAAAAACTTTAAAAAACTTAGAGTTG-3' and 5'-CAACTCTAAGTTTTTTAAAGTTTTTTGGG-3' for Y171F. Multiple mutations were set up in clones generated in the first-step mutations. The sequences of mutated Tio were confirmed using an ABI 3100 sequencer according to the manufacturer's protocol (Applied Biosystems, Darmstadt, Germany). Mutants were subcloned into herpesvirus saimiri as described previously (2, 15, 19). Tio sequences in the resulting mutant viruses and on episomes in the generated T-cell lines were amplified and again confirmed by DNA sequencing to ensure experimental integrity.
Lymphocyte culture and transformation. Immortalized lymphocyte lines 1763 Tio, 1765 Tio, and 1766 Tio (2), Jurkat T cells (E6.1, ATCC TIB-152) as well as primary lymphocytes were maintained in a 1:1 mixture of RPMI 1640 (Invitrogen, Karlsruhe, Germany) and Panserin 401 medium supplemented with 10% irradiated fetal bovine serum (Pan Biotech, Aidenbach, Germany), glutamine, and antibiotics. Human cord blood lymphocytes were obtained by selective sedimentation of erythrocytes for 45 min at 37°C in 5% dextran (molecular weight 250,000) in 150 mM NaCl. These primary cells were stimulated with 1 μg/ml phytohemagglutinin and 10 units/ml exogenous interleukin-2 (Roche Diagnostics, Mannheim, Germany) after 24 h. On the next day, the cells were infected as described (17). Cell culture supernatant was replaced step by step or supplemented by medium without interleukin-2. Cell culture densities were determined by automated cell counting (Micro Cell Counter F-300, Sysmex, Norderstedt, Germany; Z2, Beckman-Coulter, Krefeld, Germany).
PP2 assay and flow cytometry. T cells were seeded in 24-well plates at 0.5 x 106 cells/ml. PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine} and PP3 ((4-amino-7-phenylpyrazol[3,4-d]pyrimidine; Calbiochem, Merck Biosciences, Schwalbach, Germany) were dissolved in dimethyl sulfoxide at a concentration of 10 mM. Cells were treated with 10 μM PP2 or PP3 or equivalent amounts of solvent. Treated cells were counted once a day and analyzed by fluorescence-activated cell sorting on a FACScalibur flow cytometer (Becton Dickinson) using standard propidium iodide staining to detect apoptotic and dead cells, or by staining for the apoptosis marker annexin V using a kit from MedSystems Diagnostics (Vienna, Austria). The percentage of live cells was calculated as [(number of unstained cells)/(total cell number)] x 100.
Transient transfection. 293T cells were transfected with plasmid DNA using a CaCl2 transfection method. Briefly, cells were split into six-well plates. The next day each well was fed with 3.6 ml complete medium. DNA (1 to 5 μg) was diluted in 180 μl H2O and, 20 μl 2.5 M CaCl2 was added and mixed with 200 μl BES buffer (50 mM N,N-bis[2-hxdroxyethyl]-2-aminoethanesulfonic acid, 280 mM NaCl, 1.5 mM Na2HPO4, pH 6.96). This mixture was applied to the cells which were then incubated at 37°C overnight. Cells were washed twice with phosphate-buffered saline, pH 7.4, fed with 2 ml complete medium, and incubated for 16 to 24 h. Cells were harvested and frozen at –80°C or lysed for further analyses.
Immunoprecipitation, immunoblotting, and in vitro protein kinase assay. Cells were pelleted, washed, and lysed in TNE buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% NP-40) supplemented with 1 mM sodium orthovanadate (Na3VO4), 5 mM NaF, and 10 μg/ml each aprotinin and leupeptin (Sigma-Aldrich, Taufkirchen, Germany) for 20 min on ice. Lysates were cleared at 14,000 x g for 10 min, and the protein concentration of the supernatants was determined (BCA assay, Pierce, Perbio Science, Bonn, Germany). For each experiment the same amount of total protein was used; 5 μl antiserum/mg protein or 1 to 2 μg of monoclonal antibody was added for at least 1 h at 4°C to allow complex formation. Flag epitope-tagged proteins were precipitated using monoclonal antibody M2 covalently bound to agarose (Sigma, Taufkirchen, Germany). Immunoprecipitation with uncoupled antibodies was completed by incubation with protein A-Sepharose or with rabbit anti-mouse antibodies coupled to protein A-Sepharose. The immunoprecipitates were washed at least five times in TNE buffer.
For immunoblotting, cell lysates or immunoprecipitates were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane filters (Amersham Biosciences, Freiburg, Germany). Membrane filters were incubated for 1 h at room temperature in blocking buffer (phosphate-buffered saline, pH 7.4, 0.1% Tween-20, 5% nonfat dried milk powder) followed by incubation with antisera or antibody diluted in blocking buffer for 1 hour or overnight.
Anti-Tio (3) and anti-Lck (generated by immunization with a glutathione S-transferase fusion protein of human Lck amino acids 1 to 61) rabbit antisera were used at a dilution of 1/5,000. Monoclonal antibodies directed against the Flag epitope tag (clone M2, Sigma, Taufkirchen, Germany), anti-Myc tag (clone 9E10, Upstate, Biomol, Hamburg, Germany), or anti-Lck monoclonal antibodies (clone 28, Transduction Laboratories, BD Biosciences, Heidelberg, Germany) were applied at dilutions of 1/1,000 and 1/2,000, respectively. Antiphosphotyrosine monoclonal antibody PY99HRP (Santa Cruz, Heidelberg, Germany) or 4G10 (Upstate/Biomol, Hamburg, Germany) was used at a 1/10,000 dilution and milk powder was omitted. After thorough washing in phosphate-buffered saline containing 0.1% Tween 20, immunoblots were incubated with secondary antibodies coupled to horseradish peroxidase (Dako, Hamburg, Germany; Jackson Immunoresearch Laboratories, Dianova, Hamburg, Germany; Medac, Hamburg, Germany; Amersham Biosciences, Freiburg, Germany) at dilutions of 1/1,000 to 1/20,000 for 1 hour. Bands were visualized by enhanced chemoluminescence according to the manufacturer's instructions (Amersham Pharmacia Biotech, Freiburg, Germany).
For the in vitro protein kinase assay, cell lysates were adjusted to 1 ml with TNE buffer and incubated with specific antibodies at 4°C for at least 1 h and for an additional 30 min after the addition of protein A-Sepharose (Sepharose CL-4B; Amersham Biosciences, Freiburg, Germany). Immunoprecipitates were washed four times in TNE buffer, once in 10 mM Tris-HCl, pH 7.4, and once in kinase buffer containing 20 mM MOPS (morpholinepropanesulfonic acid, pH 7.0) and 5 mM MgCl2. In vitro phosphotransferase reactions were performed as described previously (7). After separation by SDS-PAGE, 32P-labeled proteins were visualized by autoradiography.
RESULTS
Growth of Tio-transformed T cells depends on Src kinase activity. Human peripheral blood lymphocytes immortalized by Tio-recombinant herpesvirus saimiri were previously found to express Tio even after prolonged cultivation (2), suggesting that the oncoprotein is required to maintain the transformed phenotype of T cells. The only cellular interaction partners of Tio identified so far are the Src family tyrosine kinases Lck, Src, and Fyn, which phosphorylate Tio upon coexpression. In addition, Tio peptides strongly bound to the SH3 domains of Lyn, Hck, and, with decreasing affinity, Lck, Fyn, Src, and Yes (3). Expression of Lck, Lyn, and Src was detectable in the long-term-cultivated Tio-expressing T-cell lines 1763 Tio, 1765 Tio, and 1766 Tio (data not shown).
To test for the biological relevance of Src kinase activity in Tio-driven T-cell immortalization, these cell lines were treated with the Src family-specific kinase inhibitor PP2 (20). Jurkat T cells were used as a negative control, as their proliferation is known to be independent of Lck, the major Src family kinase expressed in T cells. The effects of the drug were analyzed at different time points by propidium iodide staining and calculation of cell survival. Treatment with PP2 dramatically reduced the ratio of live cells in all three virus-immortalized cultures analyzed. These inhibitory effects became apparent after 18 to 40 h of treatment (Fig. 1A) and resulted in more than 90% dead cells after 6 days (Fig. 1B). In contrast, PP2 had no detectable effect on Jurkat T-cell viability even after 6 days of incubation with 10 μM of the drug. Cells treated with PP3, an inactive variant of the drug, or with solvent behaved like the untreated control. Thus, Tio-immortalized T-cell lines were dependent on the activity of Src kinases, while Jurkat T cells were not sensitive to Src kinase inhibition. These results imply a central role for the interaction between Tio and Src family kinases in the maintenance of immortalized T-cell growth.
Generation of Tio mutants. To test whether the Tio-kinase interaction is indeed the basis for the requirement of Src kinase activity and whether Tio's function also includes its substrate properties towards Src kinases, we chose to use Tio mutants deprived of their capacity to interact with or be phosphorylated by Src family kinases.
A Tio mutant expected to be deficient in binding of Src kinases was created by changing crucial residues within the SH3-binding motif, P192A and R194G. The resulting Tio mutant was called PARG (Fig. 2A). These mutations were thought to prevent Src kinase binding to Tio and possibly abolish subsequent tyrosine phosphorylation.
Tio contains four tyrosine residues, Y49, Y136, Y167, and Y171 (Fig. 2A). According to the sequence context, only one of them, Y136, has significant similarity to two tyrosine residues of Tip, Y114 and Y127 of Tip-C488 and Y72 and Y85 of Tip-C484 (Fig. 2B). These two residues of Tip-C484 are phosphorylated in the presence of Lck (21), suggesting that Tio Y136 may also be a target or substrate of the Src family kinase Lck. Tyrosine-to-phenylalanine mutations were introduced in the Tio expression plasmid in all permutations possible. The resulting 16 mutants were designated according to the residues at positions 49, 136, 167, and 171, e.g., YYYY for wild-type Tio, YFYY for phenylalanine at position 136, FYFF for phenylalanine substitution of residues 49, 167, and 171, and FFFF for the tyrosine-deficient mutant.
These expression constructs were used for transient transfection assays, and some of them were selected for the generation of recombinant herpesvirus saimiri, where StpC/Tip was replaced with a Tio expression cassette.
In vivo tyrosine phosphorylation of Tio. Wild-type and mutated Tio were transiently coexpressed along with the major T-cell Src family kinase Lck in 293T cells. Lck was the Src family kinase selected for these experiments as it is expressed in primary human T cells, the target of viral transformation. In vivo phosphorylation was analyzed by Western blots with monoclonal antiphosphotyrosine antibody PY99 (Fig. 3). Protein expression from transfected DNAs was controlled by monospecific antibodies against Lck or rabbit antiserum directed against Tio (Fig. 3, upper panel). The expression vector transfected alone served as a negative control and created no signal.
Wild-type Tio (YYYY) expressed alone was detectable with specific antiserum, but phosphorylation on tyrosine residues by endogenous Src family kinases could not be detected. In contrast, expression of Lck alone resulted in a strong autophosphorylation, confirming the functionality of the antibody used. Cotransfections of Lck with wild-type and single-tyrosine mutants of Tio show that Tio phosphorylation depends on the presence of tyrosine residue Y136. A Tio mutant in which all four tyrosines were mutated to phenylalanine (FFFF) also does not show any phosphorylation signal, confirming that a tyrosine residue within the Flag epitope tag used for all Tio expression constructs is not phosphorylated. Overexpression of Lck with Tio mutated within its SH3-binding motif (PARG) displayed a weak residual Tio phosphorylation which was interpreted as an effect of overexpression and "fly-by phosphorylation" or as an interaction between Lck and Tio different from their SH3/SH3b interaction. With respect to Lck autophosphorylation, Tio and all the tyrosine mutants showed an inhibitory effect which was abrogated by mutation of the SH3-binding motif and may correlate with a reduced expression level. Taken together, Tio was tyrosine phosphorylated only on residue 136 in the presence of cotransfected Lck in 293T cells.
Kinase interaction and phosphorylation of Tio Y136. The physical interaction between Tio and Src family kinases has been demonstrated previously (3). To identify the residues of Tio involved in this interaction, Myc-tagged Lck and Flag-tagged Tio were cotransfected in 293T cells and immunoprecipitated with appropriate monoclonal antibodies. Immunocomplexes were subjected to in vitro kinase assays and were also probed with antibodies specific for Tio and Lck (Fig. 4). Cells transfected with the expression vector alone revealed no signal. When wild-type Tio was expressed alone, the protein was precipitated by the Flag antibody (Fig. 4A), but no signal was detected after the kinase reaction, indicating that endogenous Src family kinases (like Src and Lyn) were not sufficient to create a detectable signal in this sensitive assay. Likewise, no Lck activity was precipitated by the anti-Flag monoclonal antibody.
Coexpression of Lck with Tio resulted in efficient phosphorylation of Flag-precipitated wild-type Tio-YYYY and of the mutants Tio-FYYY, Tio-YYFY, and Tio-YYYF. In contrast, Tio-YFYY, which still coprecipitated Lck, was not phosphorylated in vitro, confirming that Y136 is the only tyrosine residue phosphorylated by Lck. The negative control Tio-FFFF further demonstrated that the single tyrosine residue within the Flag epitope is not a substrate of Lck. Mutation of crucial residues within the SH3-binding domain of Tio-PARG strongly decreased binding of overexpressed Lck to Tio, leading to nearly undetectable coprecipitation of Lck and hence negligible tyrosine phosphorylation of Tio. The results of Flag-Tio precipitation were confirmed upon precipitation of Lck from aliquots of the same lysates (Fig. 4B). The amount and autophosphorylation of Lck were again reduced in all samples expressing wild-type Tio or its tyrosine mutants (Fig. 3 and 4B). These data indicated that Tio interacts directly with Lck via its SH3-binding motif and that this interaction is required for efficient phosphorylation of Tio on tyrosine residue 136.
Lymphocyte transformation by recombinant herpesvirus saimiri expressing Tio mutants. One of the unique properties of herpesvirus saimiri C488 is its ability to transform human T cells to permanent growth in vitro. We previously constructed a herpesvirus saimiri recombinant by an established cosmid recombination approach to introduce a Tio expression cassette in place of its cognate oncogenes stpC and tip. This recombinant was used to infect primary T cells. The resulting cultures continuously proliferate in the absence of interleukin-2 and express the tio oncogene.
To test whether the transforming abilities of Tio were dependent on its Src kinase binding properties and phosphorylation status, we adapted this assay system for the expression of Tio mutants in the viral background. Figure 5 shows a characteristic growth curve of cord blood lymphocyte cultures infected with recombinant viruses. Wild-type C488 herpesvirus saimiri as well as a recombinant virus with the cytomegalovirus immediate-early promoter-driven stpC and tip (M124) served as positive controls. In addition, an uninfected culture and cord blood lymphocytes infected with a control virus devoid of oncogenic sequences (331-10) were included as negative controls. Recombinant virus with wild-type Tio (YYYY), Tio with Y136 only (FYFF), or Tio under the control of its natural promoter (M134) grew independently of interleukin-2 for more than 3 months. In contrast, recombinants expressing Tio mutants unable to bind Lck (PARG) or lacking the phosphorylation site at position Y136 (FFFF and YFYY) no longer supported long-term proliferation. These results indicated that growth transformation of human T cells by these viruses was dependent on the interplay between the Tio oncoprotein and its associated Src family kinase and required phosphorylation of Tio on tyrosine residue 136.
DISCUSSION
Human T cells can be transformed to permanent growth in vitro by herpesvirus saimiri subgroup C strains encoding their own oncoproteins StpC and Tip (5) or by the Tio oncogene of herpesvirus ateles cloned into a herpesvirus saimiri backbone (2). Tio and Tip bind directly to Src family kinases and become phosphorylated on tyrosine residues (3, 7). Here we provide evidence that Y136 is the only tyrosine residue to be phosphorylated in the Tio protein and that this phosphorylation as well as the SH3-mediated binding of a Src family kinase are essential in the transformation process of primary human T cells in culture. Furthermore, the proliferation of human T lymphocytes immortalized by a Tio-recombinant virus and continuously growing for more than 2 years was blocked by inhibition of Src family kinases. As Tio was found to be constantly expressed in these cells (2), a complex between this oncoprotein and an active Src kinase also seems to play a central role in maintenance of the transformed phenotype.
The Src family kinase Lck is a key enzyme in T-cell receptor-mediated lymphocyte activation. Therefore, the T-cell-specific oncogenic effect of Tip (48) might basically be due to an upregulation of this kinase, resulting in the mimicry of an activated receptor. An increase of Lck activity was observed in the presence of Tip and assigned to the engagement of both the SH3 and kinase domains (21, 23, 30, 34). Another activating effect is suggested by the binding of a Tip Y127 phosphopeptide to the SH2 domain (4). However, studies in T-cell systems suggest a more complex regulation of Lck function by Tip, including downregulation of T-cell receptor-induced signaling at the level of Zap70, endocytosis and degradation, as well as raft recruitment of receptor complexes (8, 28, 41, 42). An influence of Tio on the activity and signaling functions of Src family kinases and their associated receptors remains to be established. However, complex formation between the oncoprotein and the SH3 domain of the kinase by itself does not seem to be sufficient in T-cell transformation by Tio-recombinant herpesvirus saimiri. Phosphorylation of the oncoprotein appears to be another essential step, as demonstrated by the Tio mutants YFYY and FFFF, which were still able to bind Lck but were no longer tyrosine phosphorylated and did not support transformation.
Phosphotyrosine is a prominent target for protein interactions. PTB and SH2 domains, which are present in various signaling proteins, bind to phosphorylated tyrosine residues with a specificity determined by the flanking amino acids. Database searches for Tio did not reveal any known phosphotyrosine motif. But the sequences flanking Y136 display similarities to the known phosphotyrosine sites of Tip (Fig. 2) (22). One of these sites in Tip (C484-Y72/C488-Y114) has been shown to be required for binding and activation of STAT1 and STAT3 (22). Similar sequences (PYLP) as well as STAT3 binding were described for StpA and StpB, the proteins encoded by the hypervariable region of herpesvirus saimiri subgroups A and B, respectively (10, 43). These proteins had previously been reported to interact with the SH2 domain of cellular Src upon phosphorylation of a YAEI/V motif (25, 33).
Tip, StpA, and StpB may therefore be considered as a scaffold, bringing together STAT3 and a Src family kinase, resulting, at least in the case of Tip and StpA, in the activation of STAT3. This model was supported by the constitutive STAT3 activation detected in T cells transformed by wild-type herpesvirus saimiri C488 and, to a lesser extent, by subgroup A strain 11 (44). However, this hypothesis was challenged by the recent observation that tyrosine residue 114 of Tip and STAT3 activity are not essential for transformation by strain C488 (24). With regard to Tio, we could not demonstrate a direct interaction between the phosphorylated oncoprotein and STAT3, although we observed constitutive phosphorylation of STAT3 in immortalized human T cells (unpublished observation). Thus, it remains to be analyzed whether STAT3 recruitment and activation are essential functions of Y136 in Tio-dependent T-cell transformation.
Peptides corresponding to the second phosphotyrosine site of Tip (C484-Y85/C488-Y127) or to the region around Y136 of Tio were recently identified as ligands for the SH2 domain of Lck (4). These data support our earlier results indicating that Tio, expressed and tyrosine phosphorylated in prokaryotic cells, binds to recombinant SH2 domains of Src kinases (3). Thus, the essential function of Y136 in Tio may be the formation of a second contact with the SH3-bound kinase, ultimately altering its activity. Such an SH2-mediated interaction seems not be required for Tip-Lck interaction, as mutation of Y127 in Tip did not interfere with transformation of human T cells by herpesvirus saimiri C488 in the presence of interleukin-2 (E. Heck and A. Ensser, personal communication). Tio would therefore display a stricter requirement for an additional SH2 interaction, which may be due to the lack of a CSKH motif interacting with the kinase domain. Alternatively, while Tip interacts specifically with Lck in T cells (49), the Src kinase associated with Tio in T cells has not been identified yet and may require different regulatory interactions.
In summary, our results indicate that growth transformation of human T cells by Tio-recombinant herpesvirus saimiri depends on the interplay between the Tio oncoprotein and a Src family kinase as well as on phosphorylation of Tio on tyrosine residue 136. Further experiments will have to address downstream signaling targets of Tio. Like Tip, Tio might affect Src kinase regulation or employ STAT3 activation. In addition, Tio might exert an StpC-related function yet to be identified. With respect to tyrosine residue 136, the search for interaction partners of phosphorylated Tio may help to identify novel signaling pathways involved in growth regulation of human T cells.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 466, TP C8), the BMBF (Interdisziplinres Zentrum für klinische Forschung Erlangen, TP B5), and the German-Israeli Foundation (674/2000).
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ABSTRACT
Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.
INTRODUCTION
Herpesvirus ateles (ateline herpesvirus 3), a monkey herpesvirus that is apathogenic in its natural host, the spider monkey (Ateles sp.), induces fatal T-cell lymphomas in other New World monkey species (16, 26, 37). Continuous T-cell lines have been established from infected animals. Infection of peripheral blood lymphocyte cultures resulted in permanently growing populations of T cells from various monkey species (18). These transforming properties are reminiscent of herpesvirus saimiri (saimiriine herpesvirus 2), the prototype of the Rhadinovirus family.
Nucleotide sequence variability in a distinct genomic locus of herpesvirus saimiri led to the classification of herpesvirus saimiri isolates into subgroups A, B, and C (12, 35, 36). While all subgroups are oncogenic in highly susceptible monkeys, other animal species and in vitro transformation experiments revealed differences in the oncogenic potential (5, 35, 46). Herpesvirus saimiri subgroup C strains, but neither subgroup A or B strains nor herpesvirus ateles isolates, are capable of transforming human T cells to permanent growth in cell culture (2, 5).
Targeted and spontaneous deletions within the herpesvirus saimiri genome indicated that proteins encoded within the subgroup-defining hypervariable region are not required for virus replication but are essential for the oncogenic phenotype of these viruses in culture and in vivo (11, 13, 14, 40). Subgroup A and B genomes encode a single saimiri transformation-associated protein, StpA and StpB, respectively (9, 25, 33, 40). In subgroup C genomes, two open reading frames were identified, encoding StpC and the tyrosine kinase-interacting protein (Tip) (6, 14).
Sequence analysis revealed the colinearity of the genomes of herpesvirus ateles and herpesvirus saimiri. A spliced gene termed tio was identified within the hypervariable region of herpesvirus ateles. The amino acid sequence encoded by this gene displayed moderate similarities to both herpesvirus saimiri subgroup C oncoproteins StpC and Tip (1, 3). Recently, we demonstrated that Tio indeed functionally replaces StpC and Tip in the process of human and monkey T-cell transformation in vitro. These data strongly suggest Tio is the oncoprotein mediating the transforming phenotype of herpesvirus ateles (2).
StpC is a 102-amino-acid protein composed of a charged N terminus followed by 18 consecutive collagen repeats and a hydrophobic C-terminal membrane anchoring sequence. The first 17 amino acids display a net negative charge and a consensus motif for tumor necrosis factor-associated factor (TRAF) binding. StpC was shown to associate with cellular Ras competing for Raf-1 binding and to activate the NF-B signaling cascade (27, 32, 45). StpC appears to be an oncoprotein, as demonstrated by the transformation of rodent fibroblast cells in culture (29) and by the development of epithelial tumors in StpC transgenic mice (31, 39), but T-cell-specific functions have not been identified so far.
Tip, the tyrosine kinase-interacting protein, interacts with the nonreceptor tyrosine kinase Lck and thereby induces various downstream signals. Its functions contributing to the control of T-cell transformation may include modulation of the T-cell receptor and its downstream signals, gamma interferon induction, and STAT factor phosphorylation (7, 8, 38, 41, 42, 47). Tip expression alone was found to be lethal in transgenic mice. However, when a conditional expression cassette was used, the mice developed fulminant T-cell lymphomas, demonstrating the T-cell specificity of Tip (48).
Tio is a 269-amino-acid protein with a negatively charged amino terminus followed by a glycine-proline-rich sequence, including some interspersed collagen-like triplets and a potential TRAF-binding motif within the amino-terminal third. This indicates a structural similarity to StpC, however, a functional correlation has not been identified. A sequence highly homologous to the Tip protein was found in the carboxy-terminal third of Tio, which corresponds to the SH3-binding motif of Tip. Coimmunoprecipitation analysis revealed that Tio interacts with nonreceptor protein tyrosine kinases of the Src family (3), suggesting that Tio may exhibit Tip-related functions in T cells.
In this study, we analyzed the relevance of the Tio-Src kinase interaction for induction and maintenance of T-cell transformation by Tio-expressing herpesvirus saimiri recombinants. We demonstrate that the Src kinase activity and Tio interaction as well as Tio phosphorylation on a single tyrosine residue are essential for the T-cell-transforming potential of Tio.
MATERIALS AND METHODS
Generation of Tio mutants and recombinant viruses. The pcDNA3-derived plasmid coding for Flag-tagged Tio (3) was modified according to the Quick Change protocol (Stratagene, Amsterdam, The Netherlands) using primer pairs 5'-CCTCCACCACAATTAGCACCTGGGCCAGCAAACC-3' and 5'-GGTTTGCTGGCCCAGGTGCTAATTGTGGTGGAGG-3' to replace P292 with A and R294 with G, 5'-CTAAAGGCGGTGGGTAATCTTCATTTTTAGAATTAATG-3' and 5'-CATTAATTCTAAAAATGAAGATTACCCACCGCCTTTAG-3' for Y49F, 5'-GCATCACGACAATATTCATCCCTTGGGAGGACGC-3' and 5'-GCGTCCTCCCAAGGGATGAATATTGTCGTGATGC-3' for Y136F, 5'-CCCTTTTAATAAATTCCCAAAAAACTATAAAAAACTTAG-3' and 5'-CTAAGTTTTTTATAGTTTTTTGGGAATTTATTAAAAGGG-3' for Y167F, and 5'-CCCAAAAAACTTTAAAAAACTTAGAGTTG-3' and 5'-CAACTCTAAGTTTTTTAAAGTTTTTTGGG-3' for Y171F. Multiple mutations were set up in clones generated in the first-step mutations. The sequences of mutated Tio were confirmed using an ABI 3100 sequencer according to the manufacturer's protocol (Applied Biosystems, Darmstadt, Germany). Mutants were subcloned into herpesvirus saimiri as described previously (2, 15, 19). Tio sequences in the resulting mutant viruses and on episomes in the generated T-cell lines were amplified and again confirmed by DNA sequencing to ensure experimental integrity.
Lymphocyte culture and transformation. Immortalized lymphocyte lines 1763 Tio, 1765 Tio, and 1766 Tio (2), Jurkat T cells (E6.1, ATCC TIB-152) as well as primary lymphocytes were maintained in a 1:1 mixture of RPMI 1640 (Invitrogen, Karlsruhe, Germany) and Panserin 401 medium supplemented with 10% irradiated fetal bovine serum (Pan Biotech, Aidenbach, Germany), glutamine, and antibiotics. Human cord blood lymphocytes were obtained by selective sedimentation of erythrocytes for 45 min at 37°C in 5% dextran (molecular weight 250,000) in 150 mM NaCl. These primary cells were stimulated with 1 μg/ml phytohemagglutinin and 10 units/ml exogenous interleukin-2 (Roche Diagnostics, Mannheim, Germany) after 24 h. On the next day, the cells were infected as described (17). Cell culture supernatant was replaced step by step or supplemented by medium without interleukin-2. Cell culture densities were determined by automated cell counting (Micro Cell Counter F-300, Sysmex, Norderstedt, Germany; Z2, Beckman-Coulter, Krefeld, Germany).
PP2 assay and flow cytometry. T cells were seeded in 24-well plates at 0.5 x 106 cells/ml. PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine} and PP3 ((4-amino-7-phenylpyrazol[3,4-d]pyrimidine; Calbiochem, Merck Biosciences, Schwalbach, Germany) were dissolved in dimethyl sulfoxide at a concentration of 10 mM. Cells were treated with 10 μM PP2 or PP3 or equivalent amounts of solvent. Treated cells were counted once a day and analyzed by fluorescence-activated cell sorting on a FACScalibur flow cytometer (Becton Dickinson) using standard propidium iodide staining to detect apoptotic and dead cells, or by staining for the apoptosis marker annexin V using a kit from MedSystems Diagnostics (Vienna, Austria). The percentage of live cells was calculated as [(number of unstained cells)/(total cell number)] x 100.
Transient transfection. 293T cells were transfected with plasmid DNA using a CaCl2 transfection method. Briefly, cells were split into six-well plates. The next day each well was fed with 3.6 ml complete medium. DNA (1 to 5 μg) was diluted in 180 μl H2O and, 20 μl 2.5 M CaCl2 was added and mixed with 200 μl BES buffer (50 mM N,N-bis[2-hxdroxyethyl]-2-aminoethanesulfonic acid, 280 mM NaCl, 1.5 mM Na2HPO4, pH 6.96). This mixture was applied to the cells which were then incubated at 37°C overnight. Cells were washed twice with phosphate-buffered saline, pH 7.4, fed with 2 ml complete medium, and incubated for 16 to 24 h. Cells were harvested and frozen at –80°C or lysed for further analyses.
Immunoprecipitation, immunoblotting, and in vitro protein kinase assay. Cells were pelleted, washed, and lysed in TNE buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% NP-40) supplemented with 1 mM sodium orthovanadate (Na3VO4), 5 mM NaF, and 10 μg/ml each aprotinin and leupeptin (Sigma-Aldrich, Taufkirchen, Germany) for 20 min on ice. Lysates were cleared at 14,000 x g for 10 min, and the protein concentration of the supernatants was determined (BCA assay, Pierce, Perbio Science, Bonn, Germany). For each experiment the same amount of total protein was used; 5 μl antiserum/mg protein or 1 to 2 μg of monoclonal antibody was added for at least 1 h at 4°C to allow complex formation. Flag epitope-tagged proteins were precipitated using monoclonal antibody M2 covalently bound to agarose (Sigma, Taufkirchen, Germany). Immunoprecipitation with uncoupled antibodies was completed by incubation with protein A-Sepharose or with rabbit anti-mouse antibodies coupled to protein A-Sepharose. The immunoprecipitates were washed at least five times in TNE buffer.
For immunoblotting, cell lysates or immunoprecipitates were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane filters (Amersham Biosciences, Freiburg, Germany). Membrane filters were incubated for 1 h at room temperature in blocking buffer (phosphate-buffered saline, pH 7.4, 0.1% Tween-20, 5% nonfat dried milk powder) followed by incubation with antisera or antibody diluted in blocking buffer for 1 hour or overnight.
Anti-Tio (3) and anti-Lck (generated by immunization with a glutathione S-transferase fusion protein of human Lck amino acids 1 to 61) rabbit antisera were used at a dilution of 1/5,000. Monoclonal antibodies directed against the Flag epitope tag (clone M2, Sigma, Taufkirchen, Germany), anti-Myc tag (clone 9E10, Upstate, Biomol, Hamburg, Germany), or anti-Lck monoclonal antibodies (clone 28, Transduction Laboratories, BD Biosciences, Heidelberg, Germany) were applied at dilutions of 1/1,000 and 1/2,000, respectively. Antiphosphotyrosine monoclonal antibody PY99HRP (Santa Cruz, Heidelberg, Germany) or 4G10 (Upstate/Biomol, Hamburg, Germany) was used at a 1/10,000 dilution and milk powder was omitted. After thorough washing in phosphate-buffered saline containing 0.1% Tween 20, immunoblots were incubated with secondary antibodies coupled to horseradish peroxidase (Dako, Hamburg, Germany; Jackson Immunoresearch Laboratories, Dianova, Hamburg, Germany; Medac, Hamburg, Germany; Amersham Biosciences, Freiburg, Germany) at dilutions of 1/1,000 to 1/20,000 for 1 hour. Bands were visualized by enhanced chemoluminescence according to the manufacturer's instructions (Amersham Pharmacia Biotech, Freiburg, Germany).
For the in vitro protein kinase assay, cell lysates were adjusted to 1 ml with TNE buffer and incubated with specific antibodies at 4°C for at least 1 h and for an additional 30 min after the addition of protein A-Sepharose (Sepharose CL-4B; Amersham Biosciences, Freiburg, Germany). Immunoprecipitates were washed four times in TNE buffer, once in 10 mM Tris-HCl, pH 7.4, and once in kinase buffer containing 20 mM MOPS (morpholinepropanesulfonic acid, pH 7.0) and 5 mM MgCl2. In vitro phosphotransferase reactions were performed as described previously (7). After separation by SDS-PAGE, 32P-labeled proteins were visualized by autoradiography.
RESULTS
Growth of Tio-transformed T cells depends on Src kinase activity. Human peripheral blood lymphocytes immortalized by Tio-recombinant herpesvirus saimiri were previously found to express Tio even after prolonged cultivation (2), suggesting that the oncoprotein is required to maintain the transformed phenotype of T cells. The only cellular interaction partners of Tio identified so far are the Src family tyrosine kinases Lck, Src, and Fyn, which phosphorylate Tio upon coexpression. In addition, Tio peptides strongly bound to the SH3 domains of Lyn, Hck, and, with decreasing affinity, Lck, Fyn, Src, and Yes (3). Expression of Lck, Lyn, and Src was detectable in the long-term-cultivated Tio-expressing T-cell lines 1763 Tio, 1765 Tio, and 1766 Tio (data not shown).
To test for the biological relevance of Src kinase activity in Tio-driven T-cell immortalization, these cell lines were treated with the Src family-specific kinase inhibitor PP2 (20). Jurkat T cells were used as a negative control, as their proliferation is known to be independent of Lck, the major Src family kinase expressed in T cells. The effects of the drug were analyzed at different time points by propidium iodide staining and calculation of cell survival. Treatment with PP2 dramatically reduced the ratio of live cells in all three virus-immortalized cultures analyzed. These inhibitory effects became apparent after 18 to 40 h of treatment (Fig. 1A) and resulted in more than 90% dead cells after 6 days (Fig. 1B). In contrast, PP2 had no detectable effect on Jurkat T-cell viability even after 6 days of incubation with 10 μM of the drug. Cells treated with PP3, an inactive variant of the drug, or with solvent behaved like the untreated control. Thus, Tio-immortalized T-cell lines were dependent on the activity of Src kinases, while Jurkat T cells were not sensitive to Src kinase inhibition. These results imply a central role for the interaction between Tio and Src family kinases in the maintenance of immortalized T-cell growth.
Generation of Tio mutants. To test whether the Tio-kinase interaction is indeed the basis for the requirement of Src kinase activity and whether Tio's function also includes its substrate properties towards Src kinases, we chose to use Tio mutants deprived of their capacity to interact with or be phosphorylated by Src family kinases.
A Tio mutant expected to be deficient in binding of Src kinases was created by changing crucial residues within the SH3-binding motif, P192A and R194G. The resulting Tio mutant was called PARG (Fig. 2A). These mutations were thought to prevent Src kinase binding to Tio and possibly abolish subsequent tyrosine phosphorylation.
Tio contains four tyrosine residues, Y49, Y136, Y167, and Y171 (Fig. 2A). According to the sequence context, only one of them, Y136, has significant similarity to two tyrosine residues of Tip, Y114 and Y127 of Tip-C488 and Y72 and Y85 of Tip-C484 (Fig. 2B). These two residues of Tip-C484 are phosphorylated in the presence of Lck (21), suggesting that Tio Y136 may also be a target or substrate of the Src family kinase Lck. Tyrosine-to-phenylalanine mutations were introduced in the Tio expression plasmid in all permutations possible. The resulting 16 mutants were designated according to the residues at positions 49, 136, 167, and 171, e.g., YYYY for wild-type Tio, YFYY for phenylalanine at position 136, FYFF for phenylalanine substitution of residues 49, 167, and 171, and FFFF for the tyrosine-deficient mutant.
These expression constructs were used for transient transfection assays, and some of them were selected for the generation of recombinant herpesvirus saimiri, where StpC/Tip was replaced with a Tio expression cassette.
In vivo tyrosine phosphorylation of Tio. Wild-type and mutated Tio were transiently coexpressed along with the major T-cell Src family kinase Lck in 293T cells. Lck was the Src family kinase selected for these experiments as it is expressed in primary human T cells, the target of viral transformation. In vivo phosphorylation was analyzed by Western blots with monoclonal antiphosphotyrosine antibody PY99 (Fig. 3). Protein expression from transfected DNAs was controlled by monospecific antibodies against Lck or rabbit antiserum directed against Tio (Fig. 3, upper panel). The expression vector transfected alone served as a negative control and created no signal.
Wild-type Tio (YYYY) expressed alone was detectable with specific antiserum, but phosphorylation on tyrosine residues by endogenous Src family kinases could not be detected. In contrast, expression of Lck alone resulted in a strong autophosphorylation, confirming the functionality of the antibody used. Cotransfections of Lck with wild-type and single-tyrosine mutants of Tio show that Tio phosphorylation depends on the presence of tyrosine residue Y136. A Tio mutant in which all four tyrosines were mutated to phenylalanine (FFFF) also does not show any phosphorylation signal, confirming that a tyrosine residue within the Flag epitope tag used for all Tio expression constructs is not phosphorylated. Overexpression of Lck with Tio mutated within its SH3-binding motif (PARG) displayed a weak residual Tio phosphorylation which was interpreted as an effect of overexpression and "fly-by phosphorylation" or as an interaction between Lck and Tio different from their SH3/SH3b interaction. With respect to Lck autophosphorylation, Tio and all the tyrosine mutants showed an inhibitory effect which was abrogated by mutation of the SH3-binding motif and may correlate with a reduced expression level. Taken together, Tio was tyrosine phosphorylated only on residue 136 in the presence of cotransfected Lck in 293T cells.
Kinase interaction and phosphorylation of Tio Y136. The physical interaction between Tio and Src family kinases has been demonstrated previously (3). To identify the residues of Tio involved in this interaction, Myc-tagged Lck and Flag-tagged Tio were cotransfected in 293T cells and immunoprecipitated with appropriate monoclonal antibodies. Immunocomplexes were subjected to in vitro kinase assays and were also probed with antibodies specific for Tio and Lck (Fig. 4). Cells transfected with the expression vector alone revealed no signal. When wild-type Tio was expressed alone, the protein was precipitated by the Flag antibody (Fig. 4A), but no signal was detected after the kinase reaction, indicating that endogenous Src family kinases (like Src and Lyn) were not sufficient to create a detectable signal in this sensitive assay. Likewise, no Lck activity was precipitated by the anti-Flag monoclonal antibody.
Coexpression of Lck with Tio resulted in efficient phosphorylation of Flag-precipitated wild-type Tio-YYYY and of the mutants Tio-FYYY, Tio-YYFY, and Tio-YYYF. In contrast, Tio-YFYY, which still coprecipitated Lck, was not phosphorylated in vitro, confirming that Y136 is the only tyrosine residue phosphorylated by Lck. The negative control Tio-FFFF further demonstrated that the single tyrosine residue within the Flag epitope is not a substrate of Lck. Mutation of crucial residues within the SH3-binding domain of Tio-PARG strongly decreased binding of overexpressed Lck to Tio, leading to nearly undetectable coprecipitation of Lck and hence negligible tyrosine phosphorylation of Tio. The results of Flag-Tio precipitation were confirmed upon precipitation of Lck from aliquots of the same lysates (Fig. 4B). The amount and autophosphorylation of Lck were again reduced in all samples expressing wild-type Tio or its tyrosine mutants (Fig. 3 and 4B). These data indicated that Tio interacts directly with Lck via its SH3-binding motif and that this interaction is required for efficient phosphorylation of Tio on tyrosine residue 136.
Lymphocyte transformation by recombinant herpesvirus saimiri expressing Tio mutants. One of the unique properties of herpesvirus saimiri C488 is its ability to transform human T cells to permanent growth in vitro. We previously constructed a herpesvirus saimiri recombinant by an established cosmid recombination approach to introduce a Tio expression cassette in place of its cognate oncogenes stpC and tip. This recombinant was used to infect primary T cells. The resulting cultures continuously proliferate in the absence of interleukin-2 and express the tio oncogene.
To test whether the transforming abilities of Tio were dependent on its Src kinase binding properties and phosphorylation status, we adapted this assay system for the expression of Tio mutants in the viral background. Figure 5 shows a characteristic growth curve of cord blood lymphocyte cultures infected with recombinant viruses. Wild-type C488 herpesvirus saimiri as well as a recombinant virus with the cytomegalovirus immediate-early promoter-driven stpC and tip (M124) served as positive controls. In addition, an uninfected culture and cord blood lymphocytes infected with a control virus devoid of oncogenic sequences (331-10) were included as negative controls. Recombinant virus with wild-type Tio (YYYY), Tio with Y136 only (FYFF), or Tio under the control of its natural promoter (M134) grew independently of interleukin-2 for more than 3 months. In contrast, recombinants expressing Tio mutants unable to bind Lck (PARG) or lacking the phosphorylation site at position Y136 (FFFF and YFYY) no longer supported long-term proliferation. These results indicated that growth transformation of human T cells by these viruses was dependent on the interplay between the Tio oncoprotein and its associated Src family kinase and required phosphorylation of Tio on tyrosine residue 136.
DISCUSSION
Human T cells can be transformed to permanent growth in vitro by herpesvirus saimiri subgroup C strains encoding their own oncoproteins StpC and Tip (5) or by the Tio oncogene of herpesvirus ateles cloned into a herpesvirus saimiri backbone (2). Tio and Tip bind directly to Src family kinases and become phosphorylated on tyrosine residues (3, 7). Here we provide evidence that Y136 is the only tyrosine residue to be phosphorylated in the Tio protein and that this phosphorylation as well as the SH3-mediated binding of a Src family kinase are essential in the transformation process of primary human T cells in culture. Furthermore, the proliferation of human T lymphocytes immortalized by a Tio-recombinant virus and continuously growing for more than 2 years was blocked by inhibition of Src family kinases. As Tio was found to be constantly expressed in these cells (2), a complex between this oncoprotein and an active Src kinase also seems to play a central role in maintenance of the transformed phenotype.
The Src family kinase Lck is a key enzyme in T-cell receptor-mediated lymphocyte activation. Therefore, the T-cell-specific oncogenic effect of Tip (48) might basically be due to an upregulation of this kinase, resulting in the mimicry of an activated receptor. An increase of Lck activity was observed in the presence of Tip and assigned to the engagement of both the SH3 and kinase domains (21, 23, 30, 34). Another activating effect is suggested by the binding of a Tip Y127 phosphopeptide to the SH2 domain (4). However, studies in T-cell systems suggest a more complex regulation of Lck function by Tip, including downregulation of T-cell receptor-induced signaling at the level of Zap70, endocytosis and degradation, as well as raft recruitment of receptor complexes (8, 28, 41, 42). An influence of Tio on the activity and signaling functions of Src family kinases and their associated receptors remains to be established. However, complex formation between the oncoprotein and the SH3 domain of the kinase by itself does not seem to be sufficient in T-cell transformation by Tio-recombinant herpesvirus saimiri. Phosphorylation of the oncoprotein appears to be another essential step, as demonstrated by the Tio mutants YFYY and FFFF, which were still able to bind Lck but were no longer tyrosine phosphorylated and did not support transformation.
Phosphotyrosine is a prominent target for protein interactions. PTB and SH2 domains, which are present in various signaling proteins, bind to phosphorylated tyrosine residues with a specificity determined by the flanking amino acids. Database searches for Tio did not reveal any known phosphotyrosine motif. But the sequences flanking Y136 display similarities to the known phosphotyrosine sites of Tip (Fig. 2) (22). One of these sites in Tip (C484-Y72/C488-Y114) has been shown to be required for binding and activation of STAT1 and STAT3 (22). Similar sequences (PYLP) as well as STAT3 binding were described for StpA and StpB, the proteins encoded by the hypervariable region of herpesvirus saimiri subgroups A and B, respectively (10, 43). These proteins had previously been reported to interact with the SH2 domain of cellular Src upon phosphorylation of a YAEI/V motif (25, 33).
Tip, StpA, and StpB may therefore be considered as a scaffold, bringing together STAT3 and a Src family kinase, resulting, at least in the case of Tip and StpA, in the activation of STAT3. This model was supported by the constitutive STAT3 activation detected in T cells transformed by wild-type herpesvirus saimiri C488 and, to a lesser extent, by subgroup A strain 11 (44). However, this hypothesis was challenged by the recent observation that tyrosine residue 114 of Tip and STAT3 activity are not essential for transformation by strain C488 (24). With regard to Tio, we could not demonstrate a direct interaction between the phosphorylated oncoprotein and STAT3, although we observed constitutive phosphorylation of STAT3 in immortalized human T cells (unpublished observation). Thus, it remains to be analyzed whether STAT3 recruitment and activation are essential functions of Y136 in Tio-dependent T-cell transformation.
Peptides corresponding to the second phosphotyrosine site of Tip (C484-Y85/C488-Y127) or to the region around Y136 of Tio were recently identified as ligands for the SH2 domain of Lck (4). These data support our earlier results indicating that Tio, expressed and tyrosine phosphorylated in prokaryotic cells, binds to recombinant SH2 domains of Src kinases (3). Thus, the essential function of Y136 in Tio may be the formation of a second contact with the SH3-bound kinase, ultimately altering its activity. Such an SH2-mediated interaction seems not be required for Tip-Lck interaction, as mutation of Y127 in Tip did not interfere with transformation of human T cells by herpesvirus saimiri C488 in the presence of interleukin-2 (E. Heck and A. Ensser, personal communication). Tio would therefore display a stricter requirement for an additional SH2 interaction, which may be due to the lack of a CSKH motif interacting with the kinase domain. Alternatively, while Tip interacts specifically with Lck in T cells (49), the Src kinase associated with Tio in T cells has not been identified yet and may require different regulatory interactions.
In summary, our results indicate that growth transformation of human T cells by Tio-recombinant herpesvirus saimiri depends on the interplay between the Tio oncoprotein and a Src family kinase as well as on phosphorylation of Tio on tyrosine residue 136. Further experiments will have to address downstream signaling targets of Tio. Like Tip, Tio might affect Src kinase regulation or employ STAT3 activation. In addition, Tio might exert an StpC-related function yet to be identified. With respect to tyrosine residue 136, the search for interaction partners of phosphorylated Tio may help to identify novel signaling pathways involved in growth regulation of human T cells.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 466, TP C8), the BMBF (Interdisziplinres Zentrum für klinische Forschung Erlangen, TP B5), and the German-Israeli Foundation (674/2000).
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