Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription
© Ammosova et al; licensee BioMed Central Ltd. 2006
Received: 27 July 2006
Accepted: 03 November 2006
Published: 03 November 2006
Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.
We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease.
Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.
The human immunodeficiency virus type 1 (HIV-1) requires host cell factors for all steps of the viral replication [1, 2]. Recently, multiple covalent modifications of viral proteins that regulate virus-host protein interactions have been described, such as phosphorylation, acetylation and ubiquitination. Phosphorylation has been reported for almost all HIV-1 accessory proteins, including Vpu , Vpr , Vif , Nef , and Rev . Transcription of HIV-1 viral genes is induced by a viral transactivator protein (Tat) [1, 2]. The activation domain of Tat (amino acids 1–48) interacts with host cell factors, whereas the positively charged RNA-binding domain (amino acids 49–57) interacts with HIV-1 transactivation response (TAR) RNA [1, 2]. In cell-free transcription assays Tat induces exclusively elongation of transcription [8, 9]. In vivo, Tat additionally induces initiation of transcription from the integrated HIV-1 promoter [10–12]. Tat stimulates formation of transcription complex containing TATA-box-binding protein (TBP) but not TBP-associated factors (TAFs), thus indicating that Tat may enhance initiation of transcription , apparently in agreement with the earlier observation that Tat binds directly to the TBP-containing basal transcription factor TFIID . Tat activates HIV-1 transcription by recruiting transcriptional co-activators that include Positive Transcription Elongation Factor b (P-TEFb), containing CDK9/cyclin T1; an RNA polymerase II C-terminal domain kinase [9, 14, 15] and histone acetyl transferases [16–18]. Whereas P-TEFb induces HIV-1 transcription from non-integrated HIV-1 template [9, 14, 15], histone acetyl transferases allow induction of integrated HIV-1 provirus [16–18]. Additional CTD kinases, including CDK2 and CDK7 were also reported to be activated by Tat and to induce functional CTD phosphorylation [19, 20]. Tat itself is a subject for covalent modifications by host cell proteins. Tat is directly acetylated at lysine 28, within the activation domain, and lysine 50, in the TAR RNA binding domain . Tat is also ubiquitinated at lysine 71 and its ubiquitination stimulates the transcriptional properties of Tat . Recently, Tat was shown to be methylated by the arginine methyltransferase, PRMT6 and the arginine methylation of Tat negatively regulated its transcriptional activity . Surprisingly, in spite of the interaction of Tat with P-TEFb and probably other kinases and its involvement in multiple protein phosphorylation reactions, the phosphorylation of HIV-1 Tat has only been reported in vitro , but not in vivo . HIV-2 Tat was reported to be phosphorylated in vivo presumably by CDK9, but this phosphorylation was not important for Tat-2 function as a transcriptional activator . We previously reported that Tat dynamically interacts with CDK2/cyclin E and is also phosphorylated by CDK2/cyclin E in vitro . This dynamic interaction greatly stimulated the activity of CDK2/cyclin E toward phosphorylation of CTD in vitro . In the present study we investigated whether Tat is phosphorylated in vivo and whether this phosphorylation has a regulatory role in Tat-activated HIV-1 transcription.
Tat is phosphorylated by CDK2 in vitroand Ser-16 and Ser-46 residues of Tat are potential phosphorylation sites
Determination of sites of phosphorylation in Tat
Peptides matching to Tat
Tat is phosphorylated in cultured cells
CDK2 phosphorylates Tat in cultured cells
Tat is phosphorylated on serine residues in vivo
Phosphorylation of S16 and S46 residues of Tat in vivo
We next investigated a possibility that Tat might be phosphorylated on S16 or S46 residues in vivo. We generated mutants of Flag-Tat in which either or both Ser residues were substituted by Ala. 293T cells were transfected with WT and mutant Tat-expressing vectors, Tat was precipitated with anti-Flag antibodies and analyzed on 15% SDS Tris-Tricine PAGE followed by PhosphoImager analysis. Expression of Tat was verified by Western blotting (Fig. 8C). While we could detect phosphorylation of WT Tat (Fig. 8C, lane 2), the Tat S16A mutant and Tat S46A mutant were about 2–3 fold less phosphorylated (Fig. 8C, middle and lower panels, lanes 3 and 4). The Tat S16,46A double mutant was even less phosphorylated (Fig. 8C, lane 5). Our results indicate that both S16 and S46 are likely to be phosphorylated in vivo.
Contribution of S16 and S46residues of Tat to HIV-1 transcription
Contribution of S16 and S46residues of Tat to the HIV-1 viral production and Tat phosphorylation in the context of the integrated HIV-1 provirus
Correlation of mutations in putative CDK2 recognition sites on Tat with disease progression in HIV infected humans
Mutation of (K/R)19 residue sequence of Tat and Sickness Status
Non-mutated Tat (K/R)19
Tat (K/R)19(T, A or G)
Our recent studies indicate that Tat's role in HIV-1 transcription is extremely complex and may not confine solely to the interaction with CDK9/cyclin T1. We have previously reported that Tat interacts directly or indirectly with host cell protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). Tat binds to protein phosphatase-1 (PP1) and this binding is important for the induction of HIV-1 transcription by Tat [31, 35]. Tat interaction with PP1 is intriguing as PP1 regulates CDK9 phosphorylation in vivo . We recently showed that Tat binds to LIS1 protein, a product of lissencephaly gene which mutations cause a severe brain malformation . LIS1 resembles the B-subunit of PP2A and interacts with the catalytic subunit of PP2A; and LIS1 expression induces HIV-1 transcription . Thus Tat might also be able to interact with PP2A, although not directly with its catalytic subunit. The current study adds more complexity to the Tat function showing that Tat might undergo phosphorylation by CDK2/cyclin E.
We hypothesized here that CDK2 affects HIV-1 transcription by phosphorylating Tat and that Tat phosphorylation might be important for HIV-1 transcription. We show here that Tat undergoes phosphorylation in vivo on serine residues, and that CDK2 is involved in this phosphorylation. Our findings also indicate that phosphorylation of Tat is important for HIV-1 transcription and the activation of integrated HIV-1 provirus. In our previous work we demonstrated that CDK2/cyclin E phosphorylates the C-terminal domain of RNA polymerase II in vitro [39–41]; that CDK2 was required for Tat-dependent transcription in vitro, and that CDK2 phosphorylates HIV-1 Tat in vitro [20, 41]. CYC202 (R-roscovitine), a pharmacological inhibitor of CDK2, efficiently inhibited replication of wild type and HAART resistant HIV-1 mutants in T-cells, monocytes and PBMCs  indicating that CDK2 activity is required for HIV-1 replication. Recently we showed that siRNA-directed against CDK2 inhibits Tat-induced HIV-1 transcription and HIV-1 viral replication . Thus our present study as well as our previous studies point to CDK2 as an important regulator of HIV-1 transcription. Until recently CDK2/cyclin E was considered to be essential for cell cycle progression and that CDK2 regulates G1/S transition by phosphorylating Rb-sequestering factors, including E2F . Recent findings challenged this role of CDK2. CDK2 knock-out mice were viable , suggesting that CDK2 is dispensable for proliferation and survival of most cell types. Also, inhibition of CDK2 activity through expression of p27 Kip1, dominant-negative CDK2, antisense oligonucleotides or siRNA did not have an effect on growth of several tumor cell lines . Therefore, not being essential for cellular viability, CDK2 might present a feasible target for anti-HIV-1 therapeutics.
Previous attempts to detect Tat phosphorylation in vivo were not successful . It is possible that low level of Tat expression or fast dephosphorylation in the cells or during sample preparation may not allow easy detection of Tat phosphorylation. For example, in the early studies Ben Berkhout and his colleagues could only detect Tat expression in COS-7 cells but not in HeLa cells [46–48]. We found that expression of Flag-tagged Tat allowed higher levels of Tat expression especially with the adeno-virus mediated delivery. Treatment with okadaic acid, which inhibits phosphatases of the PPP-family including PP1 and PP2A , significantly enhanced Tat phosphorylation (Fig. 3), suggesting that Tat may be dynamically dephosphorylated by a cellular PPP-type phosphatase. When we inhibited PP1 by over expression of the central domain of nuclear inhibitor of PP1 (NIPP1)  we did not detect changes in Tat phosphorylation (data not shown). Thus PP2A rather than PP1 is a candidate phosphatase to dephosphorylate Tat.
Our analysis showed that inhibition of CDK2 expression by siRNA substantially blocked Tat phosphorylation and prevented association of Tat with CDK2. Although these findings suggest that CDK2 might directly phosphorylate Tat, we also cannot rule out a possibility that inhibition of CDK2 reduces the activity of another kinase that in turn might be involved in Tat phosphorylation. Finding that Tat co-migrates with CDK2 on glycerol gradient and also co-precipitated with CDK2 confirms our previous observation that Tat-associated kinase activity contained CDK2 . CDK2-directed siRNA significantly reduced association of CDK2 to Tat, probably by reducing the amount of CDK2 available to interact with Tat.
We found that Tat is phosphorylated on serine residues in vivo. We previously suggested that the 16SQPK19 and K41 × L43 sequences of Tat interact with CDK2 and cyclin E respectively, and that S16 is phosphorylated by CDK2 . In the present study we found that both S16 and S46 of Tat are potential phosphorylation sites. As S46 is adjacent to the K41 × L43 sequence of Tat, it is likely that the K41 × L43 sequence participates in binding to CDK2 rather than to cyclin E, as we originally suggested. Interestingly, recombinant CDK2/cyclin E only phosphorylated full length Tat 1–72 but not the 15 amino acid peptides of Tat, 9EPWKHPGSQPKTACN23 or 37CFTTKGLGISYGRKK51 (AIDS Research and Reference Reagents Program, NIH), containing only the phosphorylation sites (data not shown), which may indicate a requirement of additional sequences of Tat for its interaction with CDK2/cycline E. Another explanation is that full length Tat creates a favorable conformation for phosphorylation by CDK2. The sequences 16SQP(K/R)19 and 46SYGR49 only partially match the CDK2 (S/T)0P1K2(K/R)3 phosphorylation motif. Although the catalytic efficiency of CDK2-cyclin A is impaired 2000-fold, when Pro1 is substituted with Ala in a short synthetic peptide substrate, physiological substrates for both CDK2-cyclin A and CDK2-cyclin E often contain phosphorylation motifs replaced with sub optimal determinants . In such sub optimal substrates phosphorylation is enhanced by a cyclin-binding motif that compensates for otherwise poor catalysis . Therefore, binding of cyclin E still might be important for efficient phosphorylation of Tat by CDK2.
Using mutation analysis, we found that S16 and S46 are equally important for activation of integrated proviral DNA. The single point mutants did not show a significant level of activation, and the double mutant Tat was completely inactive in HLM-1 cells. In contrast, mutation of S16 and S46 moderately reduced activation of HIV-1 transcription from the episomal promoter. Thus Tat S16 and S46 residues are important for transcription of a full genomic HIV-1 template containing natural chromatin structure. The effect of alanine mutation of S46 is at variance with the previously published observation that alanine mutation of S46 induces Tat-transactivation . We did not see an increase of Tat transactivation with all mutants tested. Thus we cannot explain this discrepancy. Tat was proposed to form aggregates in the nucleus . Using yeast two-hybrid system, we found no evidence that Tat forms dimers in yeast cells (not shown). Therefore, it remains to be determined why Tat should undergo phosphorylation to be fully active as a transcriptional activator. We did not detect a difference between WT and mutant Tat in ability to bind to TAR RNA (not shown). We observed an increase in the expression of untagged Tat with mutations in the S16 or Ser46 Tat residues and particular of the double mutant of Tat (Fig. 8A). An increase in Tat expression was observed earlier by Rice and Carlotti with a mutant of Tat that lacked first 36 N-terminal amino acids . Thus the amount of Tat expressed in the cells might be stringently controlled and the excess of Tat might have a negative effect on Tat transactivation. Another possibility is that Tat may need to undergo non-proteolytic ubiquitination by Hdm2 ubiquitin ligase  to be fully active as a transactivator. It is possible that Tat phosphorylation may facilitate ubiquitination of Tat by Hdm2 similar to phosphorylation-dependent ubiquitination of p53 by Hdm2 .
Analysis of Tat sequences available in the PubMed showed that Tat isolates contain from 4 to 11 serine residues. In addition to the highly conserved S16 and S46 residues, Tat contains less conserved serines at positions 23, 61, 62, 68, 70, 73, 74 and 75. Analysis of the 16SQP(K/R)19 and 46SYGR49 sequences of Tat, showed that R49 is conserved among different HIV-1 isolates. Interestingly, mutations in the (K/R)19 residue showed correlation with non-progression of HIV-1 disease. Thus (K/R)19 residue which is part of a putative CDK2 recognition site in Tat, may be important for progression of HIV-1 disease. Future study will address whether mutation of (K/R)19 residue is important for phosphorylation of Tat by CDK2 and whether mutations in this residue affect viral replication.
Taken together, our findings indicate that Tat is phosphorylated in vivo and that phosphorylation of Tat is important for the activation of integrated HIV-1 provirus. Our finding also indicates that CDK2 associates with Tat and thus is likely to phosphorylate Tat directly in vivo. Our findings open the door to the evaluation of the potential efficiency of presently available CDK2 inhibitors and specifically designed future inhibitors to disrupt CDK2-Tat association.
293T cells and COS-7 cells were purchased from ATCC (Manassas, VA). HeLa-MAGI cells , HLM-1 cells , anti-Tat rabbit polyclonal (HIV-1 BH10 Tat antiserum, ) and monoclonal (NT3 2D1.1, courtesy of Dr. Jonathan Karn) antibodies were received from the AIDS Research and Reference Reagents Program (NIH). Anti-Flag monoclonal antibodies, anti-α-tubulin antibodies, protein (G) and protein (A) agarose and okadaic acid were purchased from Sigma (Atlanta, GA). All radioactive reagents were purchased from GE Health Care Life Sciences. The 3,3'-diaminobenzidine enhanced liquid substrate system for membrane ELISA (DABM) was purchased from Sigma (St Louis, MO). Antibodies for CDK2, CDK9, cyclin T1 and cyclin T2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against RNAPII were from Babco. Anti-Brd4 and anti-HEXIM1 antibodies were a gift from Dr.Q. Zhou (University of California, Berkeley). HIV-1 Tat was expressed in Escherichia coli and purified on Aquapore RP-300 column (Applied Biosystems, Foster City, CA) by reverse-phase chromatography as we described .
Tat expression plasmid was a gift from Dr. Ben Berkhout (University of Amsterdam) . The Flag-Tat was cloned into the adeno-CMV-link vector as described below and verified by sequencing. The S16A and S46A mutations of the sequence of Tat were made according to the Quick-Change site-directed mutagenesis protocol of Stratagene, using the appropriate primers and templates. The sequences of the DNA constructs were verified by sequencing using a commercial service from Macrogen (Seoul, Korea).
CDK2/cyclin E purification
CDK2 and cyclin E were purified from lysates of Sf9 insect cells infected with baculoviruses producing CDK2 and cyclin E. Proteins were purified essentially as described earlier . Briefly, 1 ml of cells (from 250 ml of culture) was lysed with 16 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol, 10% glycerol and with PMSF), homogenized on ice and centrifuged at 45,000 g for 1 hr at 4°C. Supernatant was loaded on Mono-Q 10/10 column (Amersham, USA). Two separate cell cultures, one infected with CDK2-expressing baculovirus and the other one infected with cyclin E-expressing baculovirus were used for purification. The Mono-Q fractions containing CDK2 or cyclin E were mixed 1:1 and loaded onto Superdex column (Sephadex H200, Amersham, USA). Purity of CDK2/cyclin E was checked on 12% PAGE followed by Coomassie staining (Additional file 1). We also analyzed the Superdex fractions by immunoblotting with andti-CDK2 antibodes and assayed their enzymatic activity using histone H1 and purified Tat proteins as substrates. Fractions containing CDK2/cyclin E were concentrated using Microcon tubes (Amicon, USA).
In vitro kinase assay
CDK2 kinase assays were performed at 30°C for 30 min in kinase assay buffer (50 mM HEPES-KOH, pH 7.9, 10 mM MgCl2, 6 mM EGTA, 2.5 mM DTT) containing histone H1 or purified Tat protein, 200 μM ATP and (γ-P32)ATP. A mixture was incubated for 30 min at 30°C, reaction was stopped with 8 μl of 4× SDS buffer and resolved on 12% PAGE.
Immunoprecipitation of CDK2 and kinase assay with Tat
293T cells were transfected with CDK2-directed or non-targeting siRNA. At 48 hours after transfection cells were lysed in whole cell lysis buffer. About 50 μg of protein lysate was subjected to precipitation with anti-CDK2 rabbit antibodies (600 ng/IP) on 40 μl protein A agarose beads (50% slurry) (Sigma). As a control, rabbit preimmune serum was used. Precipitation was carried out for 2 hrs at 4°C. The beads were washed with TNN buffer, then with TTK buffer and supplemented with 20 μl of kinase mixture containing TTK buffer, 100 μM ATP, 0.5 μCi of γ-(32P)ATP and 1 μg of purified Tat. The kinase reaction was carried out for 15 min at 30°C, reaction was stopped with 8 μl of 4× SDS-loading buffer and resolved on 12% Tris-Tricine gel. The gel was stained with Coomassie blue, dried and exposed to Phosphor Imager screen.
Fractionation of cellular lysates on glycerol gradient
293T cells were infected with Adeno-Tat virus with MOI 1, and incubated overnight. The cells in 100 mm plate were lysed with 0.5 ml of whole cell lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS) supplemented with protease cocktail (Sigma) and RNasin (Amersham). Cell lysates were clarified by centrifugation for 30 min at 10,000 g and loaded on top of 10% to 30% glycerol (9 ml) gradient. Glycerol gradient buffer contained 20 mM HEPES-KOH, pH 7.9, 150 mM KCl, 200 μM EDTA
The gradient was spun in SW 41Ti rotor (Beckman) at 38,000 rpm for 18 hours. Fractions (0.5 ml) were collected through a needle inserted to the bottom of the tube and analyzed by Immunoblotting.
Tat trypsinization and chromatographic separation of peptides
Non-phosphorylated and phosphorylated Tat were resolved by 15% Tris-Tricine SDS-PAGE. A gel piece containing Tat was crushed and incubated with 1 μg of porcine trypsin (Promega, Madison, WI) in 0.1 M NaHCO3 (pH 7.9) overnight at 37°C. The extracted peptides were lyophilized, dissolved in 0.1% TCA and separated by reverse-phase chromatography on a μRPC C2/C18 ST 4.6/100 column (Amersham Pharmacia Biotech) using AKTA purifier (AmershamPharmaciaBiotech). Eluent A was 0.1% TCA in water and eluent B was 0.1% TCA in 90% acetonitrile. The gradient was 5% B for 2 column volumes (CVs), 5–50% B for 20 CVs, 50–100% B for 12 CVs and 100% B for 4 CVs. The flow rate was 0.5 ml/min.
MALDI-TOF mass spectrometry and peptide sequencing
Fractions from the HPLC separation described above were lyophilized. Multiple peptide sequences were determined in a single run by Applied Biosystems Maldi-TOF/TOF 4700 proteomics analyzer (25–30,000 resolution in reflectron mode, 5 ppm accuracy with IS, subfmole sensitivity for peptide mass fingerprints (PMF), fmole sensitivity for PSD/CID capacity of >36,000 PMF per 24 hours). Data were analyzed utilizing a local multi-processor installation of MASCOT MS PMF and MS/MS PSD, CID peptide identification software. GPS also supports an integrated MS-MS/MS mode and a chromatographic separation mode with SEQUEST-like capability.
Preparation of Adeno-Tat
The E1-deleted recombinant Ad carrying Tat was generated as previously described . Briefly, a cDNA fragment encoding the full length HIV-1 Tat protein was cloned into the plasmid pCXN was subcloned in the pAd.CMV link plasmid. The Tat-coding DNA corresponds to the HIV-1 isolate P9.3 from United Kingdom (Accession number AF324447). The DNA sequence is ATGGACTACAAGGACGACGA TGACAAAGAA TTCATGGAGC CAGTAGATCCTAGACTAGAG CCCTGGGAGC ATCCAGGAAG TCAGCCTAAG ACTGCTTGTACCCCTTGCTA TTGTAAAAAG TGTTGCTTTC ATTGCCAAGT TTGTTTCACAACAAAAGGCT TAGGCATCTC CTATGGCAGG AAGAAGCGGA GACAGCGACGAAGAGCTCCT CAAGACAGTC AGACTCATCA GGCTTCTCTA TCAAAGCAATCCCTACCCCA AACCCAGAGG GACTCGACAG GCCCGGAAGA ATCGAAGAAGGAGGTGGAGA GCAAGGCAGAGACAGATCGA TTCGATTA. It corresponds to the protein sequence of Tat containing Flag epitope at its N-terminus-MDYKDDDDKEFMEPVDPRLEPWEHPGSQPKTACTPCYCKKCCFHCQVCFTTKGLGISYGRKKRRQRRRAPQDSQTHQASLSKQSLPQTQRDSTGPEESKKEVESKAETDRFD. pAd.CMVlink and Cla I digested adenoviral DNA were co-transfected into HEK293 cells at a ratio of 3:1 by calcium phosphate precipitation to allow the recombination. The virus was purified through three rounds of plaque-purification. Viruses were replicated in HEK293 cells and were purified from a cell lysate by two rounds of CsCl density gradient centrifugation. The purified virus was desalted on a Bio-Gel P-6 desalting column (Bio-Rad Laboratories, Hercules, CA) equilibrated with PBS. The titer of the virus preparation was determined both by absorbency at 260 nm and by plaque assay. The particle to plaque forming unit ratio was less than 100. Purified viruses were suspended in PBS at the desired concentrations.
Tat phosphorylation in vivo
HeLa cells were infected with recombinant Adenovirus carrying Flag-tagged Tat prepared as we described . The purified virus had a particle to plaque forming unit (Pfu) ratio of less than 100. We added approximately 10 Pfu per cell to achieve high level of Tat expression in infected HeLa cells. At 48 hours post infection the media was changed for 1 hour to a phosphate-free DMEM media (Life Technologies, Rockville, MD) containing no serum. Then the media was changed to phosphate-free DMEM supplemented with 0.5 mCi/ml of (32P)-orthophosphate and cells were further incubated for 2 hours at 37°C. Where indicated, 1 μM okadaic acid (Sigma) was added to block cellular PPP-phosphatases. Cells were washed with PBS and lysed in whole cell lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS) supplemented with protease cocktail (Sigma). After 10 min on ice, cellular material was scraped and then centrifuged at 14,000 rpm, 4°C for 30 min. The supernatant was recovered and immediately used for immunoprecipitation. Tat was precipitated with anti-Flag monoclonal antibodies coupled to protein G agarose and with polyclonal anti-Tat antibodies coupled to protein A agarose for 2 h at 4°C in a TNN Buffer containing 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 1% NP-40. The immunoprecipitated Tat was recovered by heating for 2 min at 100°C in Tricine SDS-loading buffer, resolved on 15% Tris-Tricine SDS-PAGE  and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX). The membrane was analyzed with anti-Tat monoclonal antibodies using 3,3'-Diaminobenzidine enhancer system (Sigma) and was also exposed to Phosphor Imager screen (Packard Instruments, Wellesley, MA).
Phosphoamino acid analysis
The analysis was carried out on the Hunter thin layer peptide mapping electrophoresis system (CBS, Del Mar, CA) according to the manufacturer's recommendations. Briefly, phosphorylated Tat, prepared as described above was resolved on 15% SDS-Tris-Tricine PAGE and the gel was dried on a Whatman paper. The portion of the gel containing Tat was excised, rehydrated and treated overnight with trypsin to elute Tat. The eluted peptides were boiled in 5.7 M HCl at 110°C to liberate (32P)-labeled phospho-amino acids as described . After the hydrolysis, the sample was lyophilized and resuspended in the buffer for pH 1.9 electrophoresis also containing the phosphoamino acid standards at 0.06 mg/ml. The sample was resolved by thin-layer electrophoresis on a cellulose plate (CBS, Del Mar, CA) at pH 1.9 in the first direction and at pH 3.5 in the second direction. Cold standards were visualized by staining the plate with 0.25% ninhydrine dissolved in ethanol. Positions of labeled phosphoamino acids were analyzed with Phosphor Imager (Packard Instruments, Wellesley, MA).
Transfection and HIV-1 detection from HLM1 cells
HLM-1cells were derived from HeLa-T4+ cells integrated with one copy of HIV-1 genome containing a Tat-defective mutation. The mutation was introduced as a triple termination linker (TTL) at the first AUG of Tat gene . HLM-1 cells are negative for virus particle production HLM1 cells are negative for virus particle production. HLM1 cells can be induced to express non-infectious HIV-1 particles after transfection with Tat cDNA, or by treatment with mitogens such as TNF-α or sodium butyrate. HLM1 cells were grown in DMEM media containing 100 μg/ml of G418, plus 1% streptomycin, penicillin antibiotics and 1% L-Glutamine (Gibco/BRL). The cells grown up to 75% confluence were transfected with Tat expression vectors, including wild type Tat, S16A, S46A, and S16A/S46A mutant Tat plasmids using the calcium phosphate method. The transfected cells were washed after four hrs and fresh complete DMEM media with 10% fetal bovine was added for the remainder of the experiment. The p24 gag antigen was detected in the supernatants of transfected cells using a standard ELISA kit (Abbott).
CDK2-directed siRNA pool (M-003236-03-005) and negative control pool (D-001206-13-05) were purchased from Dharmacon (Dallas, TX). The siRNAs were transfected at final concentration of 100 nM using Lipofectamin reagent (Invitrogen) according to the manufacturer's recommendations. The siRNAs were incubated with cells for 2 days before cells were labeled with 32P or lysed for Western blotting analysis.
This work was supported by NIH Grant R21 AI 156973-01 (to S. N.), by NHLBI Research Grant UH1 HL03679 from the National Institutes of Health and The Office of Research on Minority Health. This work was also supported by grant 2 G12 RR003048 from the RCMI Program, Division of Research Infrastructure, National Center for Research Resources, NIH (to W.S.) and by NIH grants AI44357, AI43894 to FK, and Research Enhancement Fund (REF, GW) to Akos Vertes and F. Kashanchi. We thank Dr. Eric Eccleston for help with MALDI-TOF. The authors would like to thank members of Dr. Victor Gordeuk's laboratory at the Center for Sickle Cell Disease at Howard University for valuable discussions. We also thank Dr. Ajit Kumar for permission to use Hunter thin layer peptide mapping electrophoresis system. We thank Qiang Zhou (University of California, Berkeley) for the gift of ant-Brd4 and HEXIM1 antibodies.
- Brady J, Kashanchi F: Tat gets the "green" light on transcription initiation. Retrovirology. 2005, 2: 69-10.1186/1742-4690-2-69.PubMed CentralView ArticlePubMedGoogle Scholar
- Brigati C, Giacca M, Noonan DM, Albini A: HIV Tat, its TARgets and the control of viral gene expression. FEMS Microbiol Lett. 2003, 220: 57-65. 10.1016/S0378-1097(03)00067-3.View ArticlePubMedGoogle Scholar
- Coadou G, Gharbi-Benarous J, Megy S, Bertho G, Evrard-Todeschi N, Segeral E, Benarous R, Girault JP: NMR studies of the phosphorylation motif of the HIV-1 protein Vpu bound to the F-box protein beta-TrCP. Biochemistry. 2003, 42: 14741-14751. 10.1021/bi035207u.View ArticlePubMedGoogle Scholar
- Agostini I, Popov S, Hao T, Li JH, Dubrovsky L, Chaika O, Chaika N, Lewis R, Bukrinsky M: Phosphorylation of Vpr regulates HIV type 1 nuclear import and macrophage infection. AIDS Res Hum Retroviruses. 2002, 18: 283-288. 10.1089/088922202753472856.View ArticlePubMedGoogle Scholar
- Yang X, Gabuzda D: Mitogen-activated protein kinase phosphorylates and regulates the HIV-1 Vif protein. J Biol Chem. 1998, 273: 29879-29887. 10.1074/jbc.273.45.29879.View ArticlePubMedGoogle Scholar
- Cheng H, Hoxie JP, Parks WP: The conserved core of human immunodeficiency virus type 1 Nef is essential for association with Lck and for enhanced viral replication in T-lymphocytes. Virology. 1999, 264: 5-15. 10.1006/viro.1999.9937.View ArticlePubMedGoogle Scholar
- Fouts DE, True HL, Cengel KA, Celander DW: Site-specific phosphorylation of the human immunodeficiency virus type-1 Rev protein accelerates formation of an efficient RNA-binding conformation. Biochemistry. 1997, 36: 13256-13262. 10.1021/bi971551d.View ArticlePubMedGoogle Scholar
- Kim YK, Bourgeois CF, Isel C, Churcher MJ, Karn J: Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation. Mol Cell Biol. 2002, 22: 4622-4637. 10.1128/MCB.22.13.4622-4637.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu Y, Pe'ery T, Peng J, Ramanathan Y, Marshall N, Marshall T, Amendt B, Mathews MB, Price DH: Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes Dev. 1997, 11: 2622-2632.PubMed CentralView ArticlePubMedGoogle Scholar
- Raha T, Cheng SW, Green MR: HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs. PLoS Biol. 2005, 3: e44-10.1371/journal.pbio.0030044.PubMed CentralView ArticlePubMedGoogle Scholar
- Jeang KT, Berkhout B, Dropulic B: Effects of integration and replication on transcription of the HIV-1 long terminal repeat. J Biol Chem. 1993, 268: 24940-24949.PubMedGoogle Scholar
- Jeang KT, Berkhout B: Kinetics of HIV-1 long terminal repeat trans-activation. Use of intragenic ribozyme to assess rate-limiting steps. J Biol Chem. 1992, 267: 17891-17899.PubMedGoogle Scholar
- Kashanchi F, Piras G, Radonovich MF, Duvall JF, Fattaey A, Chiang CM, Roeder RG, Brady JN: Direct interaction of human TFIID with the HIV-1 transactivator tat. Nature. 1994, 367: 295-299. 10.1038/367295a0.View ArticlePubMedGoogle Scholar
- Herrmann CH, Rice AP: Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor. J Virol. 1995, 69: 1612-1620.PubMed CentralPubMedGoogle Scholar
- Yang X, Gold MO, Tang DN, Lewis DE, Aguilar-Cordova E, Rice AP, Herrmann CH: TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. Proc Natl Acad Sci U S A. 1997, 94: 12331-12336. 10.1073/pnas.94.23.12331.PubMed CentralView ArticlePubMedGoogle Scholar
- Kiernan RE, Vanhulle C, Schiltz L, Adam E, Xiao H, Maudoux F, Calomme C, Burny A, Nakatani Y, Jeang KT, Benkirane M, Van Lint C: HIV-1 tat transcriptional activity is regulated by acetylation. Embo J. 1999, 18: 6106-6118. 10.1093/emboj/18.21.6106.PubMed CentralView ArticlePubMedGoogle Scholar
- Ott M, Schnolzer M, Garnica J, Fischle W, Emiliani S, Rackwitz HR, Verdin E: Acetylation of the HIV-1 Tat protein by p300 is important for its transcriptional activity. Curr Biol. 1999, 9: 1489-1492. 10.1016/S0960-9822(00)80120-7.View ArticlePubMedGoogle Scholar
- Deng L, de la Fuente C, Fu P, Wang L, Donnelly R, Wade JD, Lambert P, Li H, Lee CG, Kashanchi F: Acetylation of HIV-1 Tat by CBP/P300 increases transcription of integrated HIV-1 genome and enhances binding to core histones. Virology. 2000, 277: 278-295. 10.1006/viro.2000.0593.View ArticlePubMedGoogle Scholar
- Cujec TP, Okamoto H, Fujinaga K, Meyer J, Chamberlin H, Morgan DO, Peterlin BM: The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II. Genes Dev. 1997, 11: 2645-2657.PubMed CentralView ArticlePubMedGoogle Scholar
- Deng L, Ammosova T, Pumfery A, Kashanchi F, Nekhai S: HIV-1 Tat interaction with RNA polymerase II C-terminal domain (CTD) and a dynamic association with CDK2 induce CTD phosphorylation and transcription from HIV-1 promoter. J Biol Chem. 2002, 277: 33922-33929. 10.1074/jbc.M111349200.View ArticlePubMedGoogle Scholar
- Bres V, Tagami H, Peloponese JM, Loret E, Jeang KT, Nakatani Y, Emiliani S, Benkirane M, Kiernan RE: Differential acetylation of Tat coordinates its interaction with the co-activators cyclin T1 and PCAF. Embo J. 2002, 21: 6811-6819. 10.1093/emboj/cdf669.PubMed CentralView ArticlePubMedGoogle Scholar
- Bres V, Kiernan RE, Linares LK, Chable-Bessia C, Plechakova O, Treand C, Emiliani S, Peloponese JM, Jeang KT, Coux O, Scheffner M, Benkirane M: A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter. Nat Cell Biol. 2003, 5: 754-761. 10.1038/ncb1023.View ArticlePubMedGoogle Scholar
- Boulanger MC, Liang C, Russell RS, Lin R, Bedford MT, Wainberg MA, Richard S: Methylation of Tat by PRMT6 regulates human immunodeficiency virus type 1 gene expression. J Virol. 2005, 79: 124-131. 10.1128/JVI.79.1.124-131.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Brand SR, Kobayashi R, Mathews MB: The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR. J Biol Chem. 1997, 272: 8388-8395. 10.1074/jbc.272.13.8388.View ArticlePubMedGoogle Scholar
- Hauber J, Bouvier M, Malim MH, Cullen BR: Phosphorylation of the rev gene product of human immunodeficiency virus type 1. J Virol. 1988, 62: 4801-4804.PubMed CentralPubMedGoogle Scholar
- Herrmann CH, Rice AP: Specific interaction of the human immunodeficiency virus Tat proteins with a cellular protein kinase. Virology. 1993, 197: 601-608. 10.1006/viro.1993.1634.View ArticlePubMedGoogle Scholar
- Koepp DM, Schaefer LK, Ye X, Keyomarsi K, Chu C, Harper JW, Elledge SJ: Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science. 2001, 294: 173-177. 10.1126/science.1065203.View ArticlePubMedGoogle Scholar
- Stevenson-Lindert LM, Fowler P, Lew J: Substrate specificity of CDK2-cyclin A. What is optimal?. J Biol Chem. 2003, 278: 50956-50960. 10.1074/jbc.M306546200.View ArticlePubMedGoogle Scholar
- http://us.expasy.org/tools/findpept.html: .
- Bennett KL, Stensballe A, Podtelejnikov AV, Moniatte M, Jensen ON: Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry. J Mass Spectrom. 2002, 37: 179-190. 10.1002/jms.271.View ArticlePubMedGoogle Scholar
- Ammosova T, Jerebtsova M, Beullens M, Voloshin Y, Ray PE, Kumar A, Bollen M, Nekhai S: Nuclear protein phosphatase-1 regulates HIV-1 transcription. J Biol Chem. 2003, 278: 32189-32194. 10.1074/jbc.M300521200.View ArticlePubMedGoogle Scholar
- Schagger H, Bentlage H, Ruitenbeek W, Pfeiffer K, Rotter S, Rother C, Bottcher-Purkl A, Lodemann E: Electrophoretic separation of multiprotein complexes from blood platelets and cell lines: technique for the analysis of diseases with defects in oxidative phosphorylation. Electrophoresis. 1996, 17: 709-714. 10.1002/elps.1150170415.View ArticlePubMedGoogle Scholar
- Ammosova T, Berro R, Kashanchi F, Nekhai S: RNA interference directed to CDK2 inhibits HIV-1 transcription. Virology. 2005, 341: 171-178. 10.1016/j.virol.2005.06.041.View ArticlePubMedGoogle Scholar
- http://www.ncbi.nlm.nih.gov/entrez: .
- Ammosova T, Jerebtsova M, Beullens M, Lesage B, Jackson A, Kashanchi F, Southerland W, Gordeuk VR, Bollen M, Nekhai S: Nuclear targeting of protein phosphatase-1 by HIV-1 TAT protein. J Biol Chem. 2005Google Scholar
- Ammosova T, Washington K, Debebe Z, Brady J, Nekhai S: Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription. Retrovirology. 2005, 2: 47-10.1186/1742-4690-2-47.PubMed CentralView ArticlePubMedGoogle Scholar
- Epie N, Ammosova T, Sapir T, Voloshin Y, Lane WS, Turner W, Reiner O, Nekhai S: HIV-1 Tat interacts with LIS1 protein. Retrovirology. 2005, 2: 6-10.1186/1742-4690-2-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Epie N, Ammosova T, Turner W, Nekhai S: Inhibition of PP2A by LIS1 increases HIV-1 gene expression. Retrovirology. 2006, 3: 65-10.1186/1742-4690-3-65.PubMed CentralView ArticlePubMedGoogle Scholar
- Nekhai S, Shukla RR, Kumar A: A human primary T-lymphocyte-derived human immunodeficiency virus type 1 Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase II and induces CAK activity. J Virol. 1997, 71: 7436-7441.PubMed CentralPubMedGoogle Scholar
- Nekhai S, Shukla RR, Fernandez A, Kumar A, Lamb NJ: Cell cycle-dependent stimulation of the HIV-1 promoter by Tat-associated CAK activator. Virology. 2000, 266: 246-256. 10.1006/viro.1999.0035.View ArticlePubMedGoogle Scholar
- Nekhai S, Zhou M, Fernandez A, Lane WS, Lamb NJ, Brady J, Kumar A: HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription. Biochem J. 2002, 364: 649-657. 10.1042/BJ20011191.PubMed CentralView ArticlePubMedGoogle Scholar
- Agbottah E, de la Fuente C, Nekhai S, Barnett A, Gianella-Borradori A, Pumfery A, Kashanchi F: Antiviral activity of CYC202 in HIV-1 infected cells. J Biol Chem. 2004Google Scholar
- Reed SI: Control of the G1/S transition. Cancer Surv. 1997, 29: 7-23.PubMedGoogle Scholar
- Malumbres M, Hunt SL, Sotillo R, Martin J, Odajima J, Martin A, Dubus P, Ortega S, Barbacid M: Driving the cell cycle to cancer. Adv Exp Med Biol. 2003, 532: 1-11.View ArticlePubMedGoogle Scholar
- Tetsu O, McCormick F: Proliferation of cancer cells despite CDK2 inhibition. Cancer Cell. 2003, 3: 233-245. 10.1016/S1535-6108(03)00053-9.View ArticlePubMedGoogle Scholar
- Verhoef K, Koper M, Berkhout B: Determination of the minimal amount of Tat activity required for human immunodeficiency virus type 1 replication. Virology. 1997, 237: 228-236. 10.1006/viro.1997.8786.View ArticlePubMedGoogle Scholar
- Verhoef K, Bauer M, Meyerhans A, Berkhout B: On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation. AIDS Res Hum Retroviruses. 1998, 14: 1553-1559.View ArticlePubMedGoogle Scholar
- Verhoef K, Berkhout B: A second-site mutation that restores replication of a Tat-defective human immunodeficiency virus. J Virol. 1999, 73: 2781-2789.PubMed CentralPubMedGoogle Scholar
- Fernandez JJ, Candenas ML, Souto ML, Trujillo MM, Norte M: Okadaic acid, useful tool for studying cellular processes. Curr Med Chem. 2002, 9: 229-262.View ArticlePubMedGoogle Scholar
- Rice AP, Carlotti F: Mutational analysis of the conserved cysteine-rich region of the human immunodeficiency virus type 1 Tat protein. J Virol. 1990, 64: 1864-1868.PubMed CentralPubMedGoogle Scholar
- Bogerd HP, Fridell RA, Blair WS, Cullen BR: Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus. J Virol. 1993, 67: 5030-5034.PubMed CentralPubMedGoogle Scholar
- Chernov MV, Bean LJ, Lerner N, Stark GR: Regulation of ubiquitination and degradation of p53 in unstressed cells through C-terminal phosphorylation. J Biol Chem. 2001, 276: 31819-31824. 10.1074/jbc.M103170200.View ArticlePubMedGoogle Scholar
- Kimpton J, Emerman M: Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol. 1992, 66: 2232-2239.PubMed CentralPubMedGoogle Scholar
- Sadaie MR, Hager GL: Induction of developmentally programmed cell death and activation of HIV by sodium butyrate. Virology. 1994, 202: 513-518. 10.1006/viro.1994.1372.View ArticlePubMedGoogle Scholar
- Hauber J, Perkins A, Heimer EP, Cullen BR: Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl Acad Sci U S A. 1987, 84: 6364-6368. 10.1073/pnas.84.18.6364.PubMed CentralView ArticlePubMedGoogle Scholar
- Rosenblatt J, De Bondt H, Jancarik J, Morgan DO, Kim SH: Purification and crystallization of human cyclin-dependent kinase 2. J Mol Biol. 1993, 230: 1317-1319. 10.1006/jmbi.1993.1248.View ArticlePubMedGoogle Scholar
- Cooper JA, Sefton BM, Hunter T: Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983, 99: 387-402.View ArticlePubMedGoogle Scholar
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