The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix
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The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4.
To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity.
Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.
KeywordsOuter Primer Relative Luciferase Activity CDK4 Kinase Activity CDK4 Region PSTAIRE Helix
The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is an essential regulator of viral replication and a critical determinant of the HTLV-induced diseases. These include the aggressive and fatal malignancy of CD4+ T-lymphocytes termed adult T-cell leukemia (ATL) [1, 2, 3]. Several lines of evidence indicate that p40 tax is the oncogene responsible for viral lymphocyte-transforming and leukemogenic properties [4, 5, 6, 7]. Mechanistically, several biochemical features of the protein can cooperate to transform, among them transcriptional stimulation of cellular signal transducers, cytokines [8, 9, 10, 11] and anti-apoptotic effectors. Tax' capacity to stimulate aneuploidy and to interfere with DNA repair  could indirectly support malignant progression. A major mechanistic explanation for the mitogenic and immortalizing effects of the Tax oncoprotein is provided by its ability to stimulate the G1- to S-phase transition in T-cells [6, 13, 14, 15].
In mammalian cells, G1-progression is controlled by the sequential activation of several cyclin-dependent kinases (CDKs), starting with CDK4, CDK6 and CDK2. Tax activates CDK4, CDK6 and CDK2 leading to phosphorylation of retinoblastoma (Rb) tumor suppressor proteins and liberation of the transcription factor E2F [6, 16]. Moreover, Tax may also induce Rb degradation  and increases cellular E2F synthesis [18, 19]. Several indirect effects of Tax and features of HTLV-infected cells may support the impact of Tax on CDK. For example, HTLV-1-infected T-cells contain increased levels of cyclin D2 [16, 20, 21], which upon binding to CDK4 forms functional holoenzyme complexes. Cyclin D2 expression is upregulated by interleukin-2 receptor (IL2-R) signals [22, 23, 24]. Tax may cooperate with interleukin-2 (IL-2) signaling either indirectly through stimulating the expression of IL-2Rα or directly by activating the cyclin D2 promoter [21, 25]. Furthermore, expression of CDK inhibitory proteins, like p18INK4C , p19INK4D and p27Kip1[16, 26] is reduced in the presence of Tax. By contrast, the inhibitory protein p21CIP1 is strongly upregulated in Tax-containing cells [20, 27]. Tax also represses the function of distinct tumor suppressor proteins which interfere with G1- to S-phase transition. These include p16INK4A, p15INK4B [26, 28, 29] and p53 [30, 31, 32, 33, 34, 35].
The protein-protein contact with the components of the cyclin D/CDK complexes provides a major explanation for the G1-phase stimulating effects of Tax. The Tax interaction with the CDK and cyclin component is direct and specific. This interaction is detectable in vitro, in transfected fibroblasts, HTLV-1-infected T-cells, and ATL-derived cultures [36, 37]. The Tax-CDK complex represents an active holoenzyme. Direct association with Tax enhances CDK4 activity. This increased kinase activity in the presence of Tax may be explained by intensified association of CDK4 and its positive cyclin regulatory subunit and by resistance of the complex to inhibition by p21CIP1 [36, 37].
To understand the molecular mechanism of the Tax-mediated CDK4 activation, the interacting domains of Tax and CDK4 were characterized. Here we show that a segment of 40 amino acids derived from the N-terminus of Tax is sufficient to bind CDK4 and cyclin D2. To define a Tax-binding domain, a series of CDK4 deletion mutants was tested in different assays. These point at two regions derived from the N- and C-terminus of CDK4 which upon deletion consistently result in reduced binding capacity. The potential of these isolated regions to interact with Tax was demonstrated by mammalian two-hybrid analysis. These experiments concurrently revealed Tax-binding at the N- and C-terminus of CDK4.
Results and discussion
Capacity of the isolated N-terminus of Tax to bind cyclin D2- and CDK4
Relevance of N- and C-terminal CDK4 regions for Tax-binding in vitro
Relevance of the N-terminal CDK4 domain for binding in vivo
Tax-binding activity of isolated CDK4 regions in vivo
To get an impression about the molecular interaction with the folded protein, a three-dimensional structure of CDK4 was calculated (Figure 5B). It resembles the structure of cdk2, which was determined from crystallized protein by X-ray diffraction . As cdk2, the predicted structure is bi-lobated, containing a β-sheet-rich N-terminal and a alpha-helix-rich C-terminal region. This structure reveals that the N- and C-terminus of CDK4 are neighbouring. Thus, it is possible that both together provide a non-continuous binding domain for Tax. The N-terminus contains the PSTAIRE helix of CDK4, which is part of the CDK's cyclin D2 binding domain. Its rotation during the activation of CDK4 is required to unblock the catalytic cleft of the kinase . Binding of Tax to this region may influence its spacial arrangement. Thus, Tax in cooperation with cyclin D2 could support formation of the active conformation and stimulate CDK4 activity by influencing the PSTAIRE helix.
The 40 N-terminal amino acids of Tax are sufficient to bind cyclin D2 and CDK4. Within CDK4 a N- and a C-terminal domain are relevant for Tax binding. These domains are neighbouring in the predicted three dimensional protein structure. Taken together, these findings suggest that Tax stimulates G1- to S-phase transition by supporting the association of CDK4 and cyclin D2. Furthermore, they support the conclusion that CDK4 activity is stimulated through conformational changes of the enzyme directly mediated by Tax.
Generation of CDK4 deletion mutants
All CDK4 deletion mutants were generated via PCR . In order to introduce the internal deletions, 16 different primers were used, two outside 28-mer oligonucleotides spanning the 5' and 3' ends of the CDK4 open reading frame (CDK4S and CDK4AS) and 14 chimeric oligonucleotides designed to carry the 5' and 3' sequences flanking the deleted regions. After three rounds of PCR with Pwo polymerase (Roche, Mannheim, Germany), the deleted clones CDK4dH30-V72, CDK4dV70-L100, CDK4dR101-L120, CDK4dM121-S150, CDK4dS150-R181, CDK4dA182-K211, CDK4dK211-D241, CDK4dV242-M275 were created. To engineer the N- terminal CDK4dM1-F31 and C-terminal CDK4dL272-E303 deletion clones, one round of PCR was performed by using an internal 5' primer or 3' primer in combination with the corresponding outside primer. To engineer the CDK4 full length construct one round of PCR was performed with the outside primers. The resulting PCR products were digested with BamHI and HindIII and ligated via these sites into the pcDNA3.1(-)/Myc-His A expression vector (Invitrogen, Karlsruhe, Germany). The resulting clones were verified by nucleotide sequencing.
Human 293T cells were kept and transfected for coimmunoprecipitations as described . Briefly, cells were lysed in buffer containing 50 mM Tris, 150 mM NaCl, 0.2% Tween 20, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml aprotinin. To immunoprecipitate Tax and associated proteins cleared protein supernatant (0.7 to 1 mg whole protein) were incubated for 1 h at 4°C with 1 μg of monoclonal Tax antibody and the immune complexes were collected by protein A-Sepharose CL4B (Pharmacia) beads (1 h at 4°C). Beads with the precipitated proteins were washed three times with lysis buffer. An aliquot of protein supernatant was taken as lysate control (40 μg whole protein). Immunoprecipitates and lysate controls were separated on gels and electro-blotted. Subsequently, membranes were incubated with 5% nonfat dry milk to block unspecific binding before reacting them with a 1: 200 dilution of monoclonal Tax antibody for 1 h at room temperature. Membranes were washed and incubated with a 1:2.500 dilution of an anti-mouse immunoglobulin G-horse-radish peroxidase conjugate (Amersham, Freiburg, Germany). Bound antibodies were visualized with an enhanced chemiluminescence detection system (Amersham) and CCD-camera. The luminescence of specific bands was quantitated from the digitalized image by using the program AIDA (raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany).
In vitrobinding and pull down assays
35S-methionine labeled CDK4 and mutants were produced in vitro with a rabbit reticolocyte-based in vitro transcription/translation system (Promega, Mannheim, Germany). To prevent the expression of the myc/his-tag, the inset plasmids were digested with HindIII prior to translation. Tax was produced in E.coli and coupled to S-protein-agarose as previousely described . For a binding assay 5–10 μl of the in vitro-translated protein was diluted in 500 μl of RIPA buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1 % Nonidet P-40, 0.5 % desoxycholat, 0.1 % sodium dodecyl sulfate). An aliquot of 10 μl was taken as an inset control. The S-protein-agarose-bound Tax protein (15 μl) was incubated with the radioactive proteins for 1 h at 4°C, washed with RIPA-buffer and recovered by boiling the beads in loading buffer. Proteins were sized on an SDS-12% polyacrylamide gel, quantitated and visualized by a phosphorimager.
TaxM1-R40 was generated via PCR, using the primers TaxM1-R40 -pet-S and TaxM1-R40 -pet-AS and plasmid pcTax  as template. Resulting PCR products and the pet 29b + vector (Novagen, Bad Soden, Germany) were digested with BamHI and HindIII and ligated. Resulting clones were verified via sequencing. Cyclin D2 and cyclin E were transfected in 293T cells and lysates were prepared as previously described . A lysate control was performed with 40 μg whole protein. Lysates containing 0.5 – 1 mg whole protein were incubated with E.coli-produced Taxwt or TaxM1-R40, coupled to Ni-NTA agarose for 1 h, washed with lysis buffer and recovered by boiling the beads in loading buffer. Proteins were sized on a 12%-SDS-PAA gel, transferred onto a nitrocellulose transfer membrane and stained with specific antibodies.
Mammalian two-hybrid assay
All constructs for mammalian two-hybrid assay were generated via PCR. The TaxM1-R40 construct PCR was performed with the primer TaxM1-R40 -M2H-S and TaxM1-R40 -M2H-AS using the plasmid pcTax as a template. For the CDK4 constructs CDK4dV70-L100 the pcDNA3.1(-)/Myc-His A construct was used as a template. The resulting PCR products were digested with KpnI and XbaI. For the other CDK4 constructs CDK4M1-V71, CDK4L100-T149, CDK4S150-K211 and CDK4V242-E303 the CDK4 full length pcDNA3.1(-)/Myc-His A construct was used as template. The resulting PCR products were digested with BamHI and XbaI. The digested products were ligated into the vectors pBind and pAct (CheckMate Mammalian two-hybrid system, Promega). The vector pG5luc contains the reporter gene (Firefly luciferase). Human 293 cells were transfected with the plasmids using Lipofectamine reagents (Invitrogen). The luciferase-assay was performed with the Dual-Luciferase reporter assay (Promega) using a microplate luminometer.
Designation for primers correspond to the plasmid names. The oligonucleotides sequences were as follows:
CDK4S, 5'-ATTTACGGATCCACCATGGCTACCTCTC-3' (outer primer);
CDK4AS, 5'-ATCCCCAAGCTTCTCCGGATTACCTTCA-3' (outer primer);
CDK4dV70-L100AS, 5'-CTTGTCCAGATATGTCCTATT GGATGCTCAAAAGC-3';
TaxM1-R40 -M2H-S, 5'-TCATCTAGAATGGCCCATTTCCCAGGGTT-3'(outer primer);
TaxM1-R40 -M2H-AS, 5'-ATTGGTACCTAGGCGGGCCGAACATAGTC-3'(outer primer);
CDK4-M2H-S, 5'-CCTTGGATCCTAATGGCTACCTCTC-3'(outer primer);
CDK4-M2H-AS, 5'-GCATTCTAGACGCCTCCGGATTACCTT-3'(outer primer);
CDK4S150-K211 -S: 5'-CCTTGGATCCTAAGTGGTGGAACAGTCAAG-3';
CDK4S150-K211 -AS: 5'-GCATTCTAGACGCCTTTCGACGAAACATCTC-3';
TaxM1-R40 -pet-S: 5'-GATCGGATCCGATGGCCCATTTCCCAGGGTT-';
TaxM1-R40 -pet-AS: 5'-CTAATTAAGCTTTAGGCGGGCCGAACATAGTCCCCCAGAGATG-3',
We thank Kerstin Haller and Ewa Blazejewska for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (SFB466-C3) and the Wilhelm Sander-Stiftung (2004.019.1).,
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