HIV-1 Tat interacts with LIS1 protein
© Epie et al; licensee BioMed Central Ltd. 2005
Received: 09 December 2004
Accepted: 07 February 2005
Published: 07 February 2005
HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization.
We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system.
Our results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation.
HIV-1 Tat protein is the viral transactivator encoded in the HIV-1 genome of infected cells [1–3]. Tat stimulates formation of full-length transcripts from the HIV-1 promoter by promoting efficient transcript elongation (reviewed in ). Tat interacts with the bulge of transactivation response (TAR) RNA, a hairpin-loop structure at the 5'-end of all nascent viral transcripts [5–7]. Tat induces elongation of HIV-1 transcription by recruiting transcriptional co-activators that include Postive Transcription Elongation Factor b (P-TEFb), an RNA polymerase II C-terminal domain kinase [8–10] and histone acetyl transferases [11–13]. Whereas P-TEFb induces HIV-1 transcription from non-integrated HIV-1 template [8–10], histone acetyl transferases allow induction of integrated HIV-1 provirus [11–13]. Tat may also increase initiation of HIV-1 transcription by enhancing phosphorylation of SP1, a transcription factor involved in the basal HIV-1 transcription . In addition to its function in HIV-1 transcription, Tat may contribute to HIV-1 pathogenesis by regulating signal transduction in endothelial cells [15, 16]; functioning as a secreted growth factor for Kaposi sarcoma and endothelial cells ; and inducing apoptosis in T-cells by binding to microtubules and delaying tubulin depolymerization [18, 19]. Tat induces apoptosis through BIM, a pro-apoptotic protein of the Bcl-2 family that antagonizes Bcl-2 anti-apoptotic proteins . The effect of Tat is similar to the effect of Taxol, a drug that stabilizes microtubules and induces apoptosis . Mutations in the glutamine-rich region of Tat protein (residues 60–72) were found to correlate with rapid progression of HIV disease, and with induction of apoptosis and binding to tubulin . We previously showed that microtubules polymerization is facilitated by LIS1 protein , a causative factor for Lissencephaly , a severe brain disorder resulted from inefficient neuronal migration during early stages of brain development . LIS1, a 45 kDa protein, contains seven repeating units called WD (Trp-Asp) repeats  that form antiparallel sheets making up a toroidal propeller structure . WD repeats containing proteins are confined to eukaryotes and participate in protein-proteins interactions . In addition to being a microtubule binding protein, LIS1 is also a subunit of platelet-activating factor acetyl hydrolase (PAF-AH) . LIS1 interacts with dynein motor, NudC and Dynactin, a complex that regulates microtubule dynamics [27, 28]. LIS1 in addition associates with Nudel , also a component of the dynein motor complex, and this interaction affects dephosphorylation of microtubules by protein phosphatase 2A (PP2A) . Thus, LIS1 may function as a scaffold that help to assemble dynein motor and serve to regulate proper microtubule dynamics.
In the present paper, we fractionated extracts of Jurkat T-cells using HIV-1 Tat as an affinity bait and RNAPII CTD activity of the Tat-associated proteins as a selection criteria. We identified by mass-spectrometry and immunoblotting components of the partially purified protein fraction and found LIS1, CDK7, cyclin H, and MAT1. We analyzed interaction of Tat with the identified individual proteins and found that Tat interacts with LIS1. We confirmed this finding by co-immunoprecipitating Tat and LIS1 from HeLa cells that were expressing Tat. And we also confirmed binding of Tat to LIS1 in a yeast two-hybrid system. Our results indicate that HIV-1 Tat interacts directly with LIS1, and therefore this interaction might contribute to the effect of Tat on microtubules formation in the cells.
LIS1, CDK7, cyclin H, and MAT1 co-purify with Tat-associated RNAPII CTD kinase activity
HIV-1 Tat interacts with WD domains of LIS1 in vitro
Tat co-immunoprecipitates with LIS1 from HeLa cellular extracts
Tat binds to LIS1 in yeast two-hybrid system
Taken together, these results indicate that LIS1 directly and specifically binds to Tat in vivo.
In this study, we show that HIV-1 Tat protein associates with LIS1 protein. LIS1, a microtubule binding protein  contains WD repeats  that are likely to participate in protein-protein interactions . LIS1 regulates microtubule dynamics by interacting with dynein motor, NudC and Dynactin [27, 28] and also with Nudel . A yeast homologue of LIS1, NudF associates with NudC to regulate dynein and microtubule dynamics [37, 38]. Thus, interaction of Tat with LIS1, a scaffold that assembles dynein motor, may affect microtubule dynamics.
We purified several candidate proteins that might interact with Tat, and found CDK7, cyclin H, MAT1 and LIS1. We expected that CDK7 might bind to Tat as previously it was shown to interact directly with Tat . In contrast, analysis of the binding of individually translated proteins showed that LIS1 and not CDK7 bound to Tat. We hypothesized that WD domain(s) of LIS1 might bind Tat, as these domains form a planar surface. Correspondingly, domains WD1, WD4, WD5 and WD7 were found to bind Tat but not the N-terminal part of LIS1 that contains coil-coil region, and which is devoid of WD domains. We analyzed whether a particular domain of Tat binds LIS1 or WD5 domain of LIS1. Full length Tat 1–72 was most efficient in binding of either LIS1 or WD5 domain of LIS1. It would be interesting to determine whether CDK7 also binds to LIS1, and whether LIS1 promotes activation of the kinase activity of CDK7 by Tat. Although LIS1 is a cytoplasmic protein, it may be required for initial assembly of a protein complex containing CDK7. Our results contrasted with the previous report in which Tat binds to purified CDK7 . We hypothesize that under our experimental conditions, excess of endogenous LIS1 present in the reticulocyte lysate might compete with interaction of Tat with CDK7. Interestingly, Gaynor an colleagues only detect specific interaction of Tat with TFIIH but not with of CDK7 or CAK alone . Therefore, it is possible that in a complex protein mixture Tat interacts with CDK7 indirectly through another protein such as LIS1.
To explore interaction of Tat and LIS1 in cultured cells, Flag-tagged Tat was expressed in HeLa cells and then immunoprecipitated with anti-Flag-antibodies. LIS1 was found to co-immunoprecipitate with Tat. Correspondingly, when LIS1 was immunoprecipitated with anti-LIS1 monoclonal antibodies, Flag-Tat was found in the immunoprecipitates. These results suggest that Tat associates with LIS1 in cultured cells. To confirm that LIS1 and Tat interact in vivo, we used yeast two-hybrid system, in which Tat was expressed as a bait and LIS1 as a prey. Again, we found that LIS1 and Tat interacted in this system. Taken together, our in vitro and in vivo results demonstrate that HIV-1 Tat binds to LIS1 and that this binding is likely to occur through one of the WD domains of LIS1.
Tat contains several functionally important regions, including the N-terminal region I (residues 1–21); cystein-rich region II (residues 22–37); core region III (residues 38–48); basic region IV (residues 49–59); glutamine-rich region V (residues 60–72); and C-terminal region VI [20, 40]. Zhou and his colleagues showed that Tat interacts with microtubules through parts of region II (residues 35–37) and region III (residue 38) . More recently, Loret and his colleagues showed that the glutamine-rich region of Tat may also interact with microtubules and promote apoptosis in T cells . In a following study which will appear in the same issue of Retrovirology, Loret and his colleagues show that Tat residues 38–72 are sufficient to enhance microtubule polymerization and that the extent of the enhancement correlates with the severity of Tat-induced apoptosis. Taken together these studies indicate that residues 35–38 of regions II and III and glutamine-rich region of Tat may interact with microtubules. These results correlate well with our finding that full length Tat binds LIS1 better than the isolate domains of Tat. Whether LIS1, a cellular structural protein and also an enzymatic subunit of PAF-AH, plays a role in Tat-induced apoptosis remained to be determined. As Tat-associated proteins include CDK7, Cyclin H, MAT1 and LIS1, it is possible that interaction of Tat with LIS1 might promote binding of CDK7 and ultimately affect viral gene expression through a direct activation of CDK7 or indirectly through activation of a down stream kinase, CDK2, by CDK7. As Tat is shuttling between nucleus and cytoplasm, its interaction with LIS1 and CDK7-containing protein complex might allow a temporary activation/modulation of the CDK7 activity. It is remained to be determined whether such interaction has an effect on Tat-induced transcription of HIV-1 genes. LIS1 may also function as an adaptor that brings HIV-1 Tat to microtubules that may release microtubules-associated BIM-1 protein and induce apoptosis . A more detailed future study will address the questions of the regulation of HIV-1 transcription and Tat-mediated apoptosis by LIS1.
Jurkat T-cells were purchased from National Cell Culture Center (CELLEX BIOSCIENCES, MN). DEAE-Sepharose (FF), SP-Sepharose (FF), Hi Trap heparin columns, [γ-32P] ATP (6000 Ci/mmol) and (35S)-labeled Methionine were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Econo-Pac CHT-II Cartridge (ceramic hydroxyapatite) was from Bio-Rad (Hercules, CA). Glutathion-agarose was from Sigma (Atlanta, GA). GST-CTD was expressed in Escherichia coli and purified as we described . The Tat expression plasmids GST-Tat (1–72), GST-Tat (1–48), GST-Tat (37–72) were obtained from AIDS Research and Reference Reagents Program (NIH), expressed in Escherichia coli and purified on Glutathione-agarose beads as described . CDK7, cyclin H and MAT1 expression vectors were kindly provided by Dr. Marcel Doreé (CNRS, Montpellier, France). Coupled transcription/translation system based on rabbit reticulocyte lysate was purchased from Ambion (Austin, TX). Protein (G) and protein (A) agarose were purchased from Sigma (Atlanta, GA).
Anti-Tat rabbit polyclonal (HIV-1 BH10 Tat antiserum) and monoclonal (NT3 2D1.1) antibodies were received from AIDS Research and Reference Reagents Program (NIH). Anti-Flag antibodies were purchased from Sigma (Atlanta, GA). Polyclonal antibodies to CDK7, and PSTAIRE were purchased from Santa Cruz Biochemical (Santa Cruz, CA). Polyclonal antibody to CDK9 (PITALRE) were purchased from Biodesign Company (Saco, ME). Monoclonal antibodies for LIS1 were as described .
Tat-associated CTD kinase assay
Tat-associated kinase activity was assayed as described previously . Briefly, portions of eluted fractions (about 1/1000 of the total amount) from each chromatography column were incubated with 10 μg of GST-Tat (1–72) immobilized on glutathione-agarose beads for 1 hour at 4°C. The beads were washed with the buffer B containing 20 mM HEPES (pH 7.9), 250 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF and 10 μg/ml aprotinin, followed by washing with the kinase buffer (50 mM HEPES (pH 7.9), 10 mM MgCl2, 6 mM EGTA and 2.5 mM dithiothreitol). Tat-associated CTD kinase activity was assayed by incubating the kinase-bound beads with 100 ng GST-CTD in kinase buffer containing 50 μM ATP and 10 μCi of ( 32p)ATP for 10 min at room temperature. Phosphorylated GST-CTD was resolved on 10% SDS-PAGE and subjected to autoradiography and quantification with PhosphorImager Storm 860 (Molecular Dynamics).
Purification of Tat-associated CTD kinase
Purification of Tat-associated CTD kinase from Jurkat T-cells was carried as previously described . Briefly, 100 liters of Jurkat T cell culture at concentration of 5 × 105 cells/ml were centrifuged, washed and Dounce-homogenized in Buffer A (50 mM HEPES [pH 7.9], 5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin and 10% glycerol) supplemented with 0.1% NP-40. The whole cell extract was prepared and fractionated by ammonium sulfate precipitation. Ammonium sulfate was added to 10% saturation to extract nuclei. After centrifugation, the supernatant, containing approximately 10 g of protein, was further fractionated with ammonium sulfate added to 20%, 40% and 80% saturation. The 40% ammonium sulfate fraction (about 3.5 g of protein) was found to contain the major part of Tat-associated CTD kinase activity. This fraction was diluted with Buffer A until the conductivity was equivalent to 50 mM KCl and then loaded on a DEAE-Sepharose column (about 500 mg of protein per 50 ml column). The column was eluted with a linear gradient of KCl (0.1 to 1 M) in Buffer A. Fractions were assayed for Tat-associated CTD kinase activity as described above. A peak of Tat-associated CTD kinase activity was collected, diluted with Buffer A until conductivity was equivalent to 50 mM KCl and loaded on a 10 ml SP-Sepharose column which was eluted with linear gradient of KCl (0.1 to 1 M) in Buffer A. A flow-through fraction containing Tat-associated CTD kinase activity was further fractionated on Hi Trap heparin columns (1 ml, three in series). Fractions were collected and analyzed for the Tat-associated CTD-kinase activity as described above, as well as by immunoblotting. Fractions containing Tat-associated CTD kinase activity TTK were resolved on 12% SDS-PAGE (20 × 20 cm) stained with colloidal Coumassie Blue and subjected to protein microsequencing.
NanoLC ion trap mass spectrometry and peptide sequencing
The procedure for peptide sequencing was performed as described previously. Protein bands visible after colloidal Coomassie blue staining and corresponding to the peak of CTD hyperphosphorylating activity after the heparin-agarose column were subjected to in-gel reduction, carboxyamidomethylation and tryptic digestion (Promega, Madison, WI). Multiple peptide sequences were determined in a single run by microcapillary reverse-phase chromatography directly coupled to a Finnigan LCQ quadrupole ion trap mass spectrometer equipped with a custom nanoelectrospray source. The column was packed in-house with 5 cm of C18 support into a New Objective one-piece 75 um I.D. column terminating in a 15 μm tip. Flow rate was 190 nanoliters/min. The ion trap was programmed to acquire successive sets of three scan modes consisting of full scan MS over alternating ranges of 395–800 m/z or 800–1300 m/z, followed by two data dependent scans on the most abundant ion in those full scans. These data dependent scans allowed the automatic acquisition of a high resolution (zoom) scan to determine charge state and exact mass, and MS/MS spectra for peptide sequence information. MS/MS spectra were acquired with a relative collision energy of 30%, an isolation width of 2.5 Dalton and recurring ions dynamically excluded. Interpretation of the resulting MS/MS spectra of the peptides was facilitated by programs developed in the Harvard Microchemistry Facility and by database correlation with the algorithm SyQuest .
In vitro proteins synthesis
Proteins were transcribed/translated as described previously . Briefly, the CDK7, cyclin H and MAT1, LIS1 and different domains of LIS1 were transcribed/translated in a coupled rabbit reticulocyte system according to manufacturer recommendations (Ambion, Austin, TX). Proteins were resolved on 12% SDS-PAGE. The gel was treated with Amplify solution (Amersham Pharmacia Biotech, Piscataway, NJ), dried and exposed to X-ray film with intensifying screen at -70°C.
Co-immunoprecipitation and Western blot
HeLa cells were infected with adenovirus vector expressing Flag-tagged Tat protein as we previously described . HeLa whole cell extracts were prepared as described previously . Cell extracts were also prepared from non-infected HeLa cells and used as a control. About 100 μg of whole cell extract was supplemented with 5 μg of anti-Flag or anti LIS1 antibodies. Then protein G-agarose beads preblocked with 5% BSA and suspended in TNN buffer (50 mM Tris-HCl (pH 7.5), 0.5% NP-40, 150 mM NaCl) buffer were added and the reaction was incubated in TNN buffer at 4°C for 2 h with rocking. The beads were precipitated and washed once with TNN buffer and once with the kinase buffer (50 mM HEPES-KOH (pH-7.9), 10 mM MgCl2, 6 mM EGTA, 2.5 mM DTT). The pellet was then resuspended in a 30 μl of 1X SDS loading buffer (4% SDS, 10% glycerol, 5% 2-mecarpthaethanol, 0.002% bromophenol blue) and heated at 90°C for 3 minutes. The proteins were resolved on SDS Tris-Tricine PAGE, 10%, to detect LIS1, or 12%, to detect Tat, and immunoblotted with anti-LIS1 or anti-Flag antibodies.
Yeast two-hybrid system
The parent yeast cells EGY48 (LexA 2H) genotype (MATα, ura3, his3, tryp1, LexAop (x6) -LEU2), auxotrophic for tryptophan (Trp), uracil (Ura), histidine (His), with LEU2 as a reporter gene. Yeast were transformed by electroporation as follow. One colony of the yeast cells was resuspended into 10 ml of appropriate selective media and grown at 30°C overnight. Cells were collected at 3000 rpm for 10 min, washed twice with HEPES/Sorbitol (20 mM HEPES pH 7.9, 1 M Sorbitol), resuspended in 200 μl of HEPES/Sorbitol and supplemented with 1 μg of a plasmid DNA. The mixture was pulsed with 2500 V in 0.4 cm cuvette, then 1 ml of appropriate selective media was added and cells were shaken at 30°C for 2 hours. The cells were collected by centrifugation, resuspended into 250 μl of HEPES/Sorbitol and plated on appropriate selective plates. EGY48 cells were transformed with pSH18–34 vector containing Lac Z reporter under the control of LexAop(x8) and also URA3 and amp r genes as selection markers. The transformed yeast cells (EGY48-lacZ) were selected on Uracyl deficient media. HIV-1 Tat first exon was subcloned into pLexA in frame with the LexA(1–202), the DNA binding domain to create the bait vector (pLexA-Tat). LIS1 was subcloned into pJG 4–5 (amp r ) in frame with the acid activation domain to create pJG-LIS1 carrying hemagglutinin (HA) tag (Trp selectable marker). The EGY48-lacZ yeast cells were transformed with pLexA-Tat vector, and selected for growth on uracyl and histidine deficient media. The Tat expressing yeast cell growing on Uracyl, Histidine deficient plates were then transformed with pJG-LIS1. To detect interaction between Tat and LIS1 interaction, yeast cells were plated on galactose/raffinose-containing plates to allow expression of Tat and LIS1, and production of β-galactosidase was visualized with 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) substrate.
This work was supported by NIH Grants AI 156973-01 and AI 056973-01S1, and by NHLBI Research Grant UH1 HL03679 from the National Institutes of Health and The Office of Research on Minority Health. The work was also supported in part by "The Nella and Leon Benoziyo Center for Neurological Diseases". O.R. is an Incumbent of the Berstein-Mason professorial chair of Neurochemistry. The authors would like to thank Dr. Victor Gordeuk, the director of the Research Scientist Program of Howard University for his continuous support and members of his laboratory at the Center for Sickle Cell Disease of Howard University for valuable discussions.
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