Inhibition of PP2A by LIS1 increases HIV-1 gene expression
© Epie et al; licensee BioMed Central Ltd. 2006
Received: 21 March 2006
Accepted: 02 October 2006
Published: 02 October 2006
Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR.
Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity.
Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A.
Tat protein is a transcriptional activator encoded in the genome of HIV-1 (reviewed in ). Tat binds to a transactivation response (TAR) RNA  and activates HIV-1 transcription by recruiting transcriptional co-activators that include Positive Transcription Elongation Factor b and histone acetyl transferases [2–4]. In addition to its function in HIV-1 transcription, Tat also interacts with a number of cellular factors thus affecting host cellular functions [5, 6]. In T cells, Tat causes apoptosis by binding to microtubules and affecting microtubule formation . Tat also causes apoptosis in neurons apparently by changing polarity of the neuronal membranes [8, 9]. Previously, we reported that Tat binds to LIS1 . LIS1 is a microtubule binding protein and its mutation causes Lissencephaly, a severe brain malformation . Lissencephaly is caused by abnormal neuronal migration during brain development . LIS1 is 45 kD protein that contains seven WD repeats and an N terminal domain devoid of the repeats. The WD repeats-containing proteins fold into a beta propeller structure that participates in protein-protein interaction in cells . The diverse family of WD40 proteins includes B-subunits of protein phosphatase 2A (PP2A). PP2A is a major serine/threonine phosphatase found mainly in the nucleus but also present in the cytoplasm . PP2A catalytic subunit associates with the A subunit to form the core enzyme, and with the A and B subunits to form the holoenzyme . The B subunits are diversified and represented by a variety of proteins ranging from 45 kD to 55 kD [15–17]. B subunits target PP2A to different locations within the cell [18–20]. PP2A was reported to affect HIV-1 transcription both positively and negatively. Deregulation of cellular enzymatic activity of PP2A inhibited Tat-induced HIV-1 transcription [21, 22]. Expression of the catalytic subunit of PP2A enhanced activation of HIV-1 promoter by phorbol myristate acetate (PMA), whereas inhibition of PP2A by okadaic acid and by fostriecin prevented activation of HIV-1 promoter . In contrast, inhibition of PP2A was shown to induce phosphorylation of Sp1 and upregulate HIV-1 transcription .
In this report, we investigate the effect of LIS1, full length or its isolated domains, on Tat mediated HIV-1 transcription in 293 cells. We compared the effect of LIS1 with the effect of okadaic acid, a known inhibitor of PP2A. We also analyzed the effect of LIS1 on strong viral cytomegalovirus (CMV) promoter and a strong cellular phosphoglycerate kinase (PGK) promoter. Observing similar effects of LIS1 and okadaic acid, we also analyzed the effect of LIS1 on the activity of PP2A in vitro. Our results presented here point to LIS1 as a yet unrecognized regulator of PP2A that may contribute to the regulation of HIV-1 transcription.
LIS1 induces HIV-1 transcription
WD5 domain of LIS1 upregulates Tat mediated transcription
The WD5 domain of LIS1 inhibits phosphorylase-phosphatase activity of PP2A
Binding of Tat to LIS1 does not affect the inhibition of PP2A by LIS1
Taken together, our results show that LIS1 upregulates HIV-1 Tat mediated transcription and that this upregulation could be due to the inhibition or modulation of PP2A activity by LIS1.
Our results presented here show that LIS1 upregulates HIV-1 transcription possibly by inhibiting PP2A. We demonstrate that the WD domains but not the N terminal domain of LIS1 are involved in both upregulation of transcription and PP2A inhibition.
LIS1, a microtubule binding protein  regulates microtubule dynamics by interacting with dynein motor, NudC and Dynactin [29, 30] and also with Nudel . A yeast homologue of LIS1, NudF associates with NudC to regulate dynein and microtubule dynamics [32, 33]. Lissencephaly is a neuronal disease caused by a severe mutation in the LIS1 gene. Interestingly, HIV-1-associated dementia is prevalent in the patients with AIDS. Whether there is a connection between deregulation of LIS1 function and development of dementia is not yet known, but obviously this is an intriguing possibility.
PP1 was a generous gift from Dr. Bollen (Catholic University of Leuven, Belgium). PP2A was purchased from Upstate (Chicago, IL). Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals. Anti-Flag and anti-a-tubulin antibodies were from Sigma.
The HIV-1 reporter contained HIV-1 LTR (-138 to +82) followed by a nuclear localization signal (NLS) and the LacZ reporter gene (courtesy of Dr. Michael Emmerman, Fred Hutchinson Cancer Institute, Seattle, WA). It expresses NLS-tagged β-galactosidase under the control of HIV-1 LTR (16). The HIV-1 reporter plasmid without TAR contained a deletion of +19 to +87 nucleotides of LTR introduced by restriction digestion with BglII. The Tat expression plasmid was a gift from Dr. Ben Berkhout (University of Amsterdam) (17). The CMV-EGFP cloned into the Adenovirus shuttle vector was a gift from Dr. Marina Jerebtsova (Children's National Medical Center). The SIN vector containing phosphoglycerol kinase (PGK) promoter followed by EGFP  was a gift from Dr. John Tisdale (NIDDK, NIH). The PP2A Bγ expression vector  was a gift from Dr. Stefan Strack (University of Iowa).
Expression and purification of WD5 and N terminal of LIS1
The DNA sequences encoding each of the seven WD domains and the amino terminal domain of LIS1 were subcloned into a plasmid carrying T7 promoter upstream of the multiple cloning site and a myc tag and ampr markers. These vectors were created at the laboratory of Dr. Orly Reiner (The Weizmann Institute of Science, Israel) and were kindly given to us. The DNA was transformed into E. coli BL21 SI cells. The cells were grown to mid log phase, and synthesis of recombinant proteins was induced by the addition of NaCl to a final concentration of 0.3M for 18 hours according to the recommendation of manufacturer (Invitrogen). The cells were lysed by sonication in a buffer A (10 mM Tris-HCl (pH 7.8), 50 mM NaCl, 1 mM EDTA 1 mM PMSF, 20% glycerol) and inclusion bodies were recovered by centrifugation. Inclusion bodies were dissolved in the buffer A containing additionally 6M urea and proteins were concentrated on microcone spinning tubes (Millipore, Billerica, MA). The recombinant proteins were dialyzed against PBS before usage.
Cell culture and transfection
Cells were maintained in Dubelco Modified Eagles Medium (DMEM) supplemented with 10% FBS and 0.1%penicillin/streptomycin. HEK293 cells were subcultured 24 hrs prior to transfection to achieve 60% confluence on the day of transfection. Transfections were carried out in 96 well plates and in some experiments in 6 well plates. Transfections were performed by calcium phosphate precipitation as previously described .
Subcloning of pCI-LIS
LIS1 gene was subcloned from pAGA2 vector  to pCI-Neo eukaryotic expression vector (Promega, Madison, WI). The pAGA2-LIS1 plasmid was digested with EcoR1 and Sal1 to extract LIS1 fragment. The LIS1-containig DNA fragment was purified on the agarose gel and ligated into the pCI-Neo digested with EcoR1 and Sal1. The resulting plasmid pCI-LIS was checked by restriction digestion with EcoR1 and Sal1 to visualize ligation products on an agarose gel and also by sequencing with T7 and T3 primers.
HEK 293 cells transfected with HIV-1 LTR-LacZ and HIV-1 Tat expression vectors were lyzed and the level of transcription from the HIV-1 LTR was determined by measuring the β-galactosidase activity as previously described . Briefly, cells were washed with phosphate-buffered saline (PBS) and lysed for 20 min at room temperature in 50 μl of lysis buffer, containing 20 mM HEPES at pH 7.9, 0.1% Nonidet P-40, and 5 mM EDTA. Subsequently, 100 μl of o-nitrophenyl-β-D-galactopyranoside (ONPG) solution (72 mM Na2PO4 at pH 7.5, 1 mg/ml ONPG, 12 mM MgCl2, 180 mM 2-mercaptoethanol) was added and incubated at room temperature until a yellow color developed. The reaction was stopped by addition of 100 μl of 1 M Na2CO3. The 96-well plates were analyzed in a micro plate reader at 414 nm (Lab Systems Multiscan MS). The β-galactotosidase units were calculated using a linear graph plotted from optical density (OD) readings of the standard.
293T cells transfected in 96-well plate were lysed in 50 μl of lysis buffer per well (20 mM HEPES at pH 7.9, 0.1% Nonidet P-40, and 5 mM EDTA), then supplemented with 150 μl of PBS and transferred to fluorescent-compatible 96-well plate. The GFP fluorescence was measured at 480 nm excitation and 510 nm emission on Luminescence Spectrometer LS50B (Perkin-Elmer) equipped with the robotic 96-well scanner.
Cells transfected with various vectors were washed 3 times with PBS and then lyzed with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1% NP-40, 0.1% SDS and protease inhibitor cocktail (Sigma). The control cell extracts were prepared from mock-transfected 293T cells. About 5 μg whole cell extract proteins were resuspended in a 30 μl of 1× 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 10% SDS Polyacrylamide gel electrophoresis (PAGE) and immunoblotted with anti-LIS1 or anti tubulin antibodies
Phosphorylase-a was prepared as previously described . Approximately 0.2 nmol of phosphorylase-a was used as a substrate for PP1 or PP2A. The phosphorylase phosphatase assay was carried out for 10 min in a buffer containing 50 mM glycylglycine at pH 7.4, 0.5 mM dithiothreitol, and 5 mM β-mercaptoethanol as described . Where indicated, prior to the phosphorylase phosphatase assay, the samples were trypsinized to generate free, active catalytic subunit of PP1.
This work was supported by NIH Research Grant # UH1 HL03679 funded by National Heart, Lung and Blood Institute and The Office of Research on Minority Health; and by grant AI056973. The pGEM2Tat bacterial expression vector was provided by Richard Gaynor and obtained through the NIH AIDS Research and Reference Reagents Program, Division of AIDS, NIAID, NIH. We would like to thank Dr. Victor Gordeuk and members of the Research Scientist Laboratory for their suggestions and discussion. We would like to thank Orly Reiner (The Weizmann Institute of Science, Israel), Mathieu Bollen and Monique Beullens (Catholic University, Leuven, Belgium), Marina Jerebtsova (Children's National Medical Center), John Tisdale (NIDDK, NIH) and Stefan Strack (University of Iowa) for the gift of reagents.
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