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The Mycobacterium tuberculosis Protein Kinase K Modulates Activation of Transcription from the Promoter of Mycobacterial Monooxygenase Operon through Phosphorylation of the Transcriptional Regulator VirS

2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês

10.1074/jbc.m808705200

ISSN

1083-351X

Autores

Pawan Kumar, Devanand Kumar, Amit Parikh, Dimple Rananaware, Meetu Gupta, Yogendra Singh, Vinay Kumar Nandicoori,

Tópico(s)

Biochemical and Molecular Research

Resumo

Mycobacterium tuberculosis encodes for 11 eukaryotic-like serine/threonine protein kinases. Genetic and biochemical studies show that these kinases regulate various cellular processes including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion and the expression, and/or activity of transcription factors. PknK is the largest predicted serine/threonine protein kinase in M. tuberculosis. Here, we have cloned, overexpressed, and purified protein kinase K (PknK) to near homogeneity and show that its ability to phosphorylate proteins is dependent on the invariant lysine (Lys55), and on two conserved threonine residues present in its activation loop. Despite being devoid of any apparent transmembrane domain, PknK is localized to the cell wall fraction, suggesting probable anchoring of the kinase to the cell membrane region. The pknK gene is located in the vicinity of the virS gene, which is known to regulate the expression of the mycobacterial monooxygenase (mymA) operon. We report here for the first time that VirS is in fact a substrate of PknK. In addition, four of the proteins encoded by mymA operon are also found to be substrates of PknK. Results show that VirS is a bona fide substrate of PknK in vivo, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA. Reporter assays reveal that PknK modulates VirS-mediated stimulation of transcription from the mym promoter. These findings suggest that the expression of mymA operon genes is regulated through PknK-mediated phosphorylation of VirS. Mycobacterium tuberculosis encodes for 11 eukaryotic-like serine/threonine protein kinases. Genetic and biochemical studies show that these kinases regulate various cellular processes including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion and the expression, and/or activity of transcription factors. PknK is the largest predicted serine/threonine protein kinase in M. tuberculosis. Here, we have cloned, overexpressed, and purified protein kinase K (PknK) to near homogeneity and show that its ability to phosphorylate proteins is dependent on the invariant lysine (Lys55), and on two conserved threonine residues present in its activation loop. Despite being devoid of any apparent transmembrane domain, PknK is localized to the cell wall fraction, suggesting probable anchoring of the kinase to the cell membrane region. The pknK gene is located in the vicinity of the virS gene, which is known to regulate the expression of the mycobacterial monooxygenase (mymA) operon. We report here for the first time that VirS is in fact a substrate of PknK. In addition, four of the proteins encoded by mymA operon are also found to be substrates of PknK. Results show that VirS is a bona fide substrate of PknK in vivo, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA. Reporter assays reveal that PknK modulates VirS-mediated stimulation of transcription from the mym promoter. These findings suggest that the expression of mymA operon genes is regulated through PknK-mediated phosphorylation of VirS. Cell signaling is a process by which environmental signals are transmitted to cells, ultimately resulting in changes in gene expression and activity. One of the major mechanisms by which this takes place is by the reversible phosphorylation of cellular proteins. Protein phosphorylation in prokaryotes plays a regulatory role in events as diverse as chemotaxis, bacteriophage infection, nutrient uptake, and gene transcription (1.Av-Gay Y. Everett M. Trends Microbiol. 2000; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). The enzymes involved in these regulatory functions are either protein histidine kinases, phosphotransferases, or protein serine kinases. Analysis of the Mycobacterium tuberculosis genome sequence suggests the presence of 11 eukaryotic-like serine/threonine protein kinases, two tyrosine protein phosphatases, and one serine/threonine protein phosphatase (2.Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar). Nine of these kinases contain a putative transmembrane domain, suggesting their localization to the cell membrane. To date, nine of these kinases have been biochemically characterized. They are PknA, PknB, PknD, PknE, PknF, PknG, PknH, PknI, and PknL (3.Chaba R. Raje M. Chakraborti P.K. Eur. J. Biochem. 2002; 269: 1078-1085Crossref PubMed Scopus (89) Google Scholar, 4.Av-Gay Y. Jamil S. Drews S.J. Infect. Immun. 1999; 67: 5676-5682Crossref PubMed Google Scholar, 5.Peirs P. Parmentier B. De Wit L. Content J. FEMS Microbiol. Lett. 2000; 188: 135-139Crossref PubMed Google Scholar, 6.Molle V. Girard-Blanc C. Kremer L. Doublet P. Cozzone A.J. Prost J.F. Biochem. Biophys. Res. Commun. 2003; 308: 820-825Crossref PubMed Scopus (42) Google Scholar, 7.Koul A. Choidas A. Tyagi A.K. Drlica K. Singh Y. Ullrich A. Microbiology. 2001; 147: 2307-2314Crossref PubMed Scopus (90) Google Scholar, 8.Molle V. Kremer L. Girard-Blanc C. Besra G.S. Cozzone A.J. Prost J.F. Biochemistry. 2003; 42: 15300-15309Crossref PubMed Scopus (118) Google Scholar, 9.Gopalaswamy R. Narayanan P.R. Narayanan S. Protein Expression Purif. 2004; 36: 82-89Crossref PubMed Scopus (33) Google Scholar, 10.Lakshminarayan H. Narayanan S. Bach H. Sundaram K.G. Av-Gay Y. Protein Expression Purif. 2008; 58: 309-317Crossref PubMed Scopus (20) Google Scholar). Although much work remains to be done toward understanding the role of the STPKs in M. tuberculosis biology, recent reports have established the role of these kinases in modulating cell shape and morphology, glucose transport, glutamine transport, glutamate metabolism, and regulation of the expression/activity of transcription factors (8.Molle V. Kremer L. Girard-Blanc C. Besra G.S. Cozzone A.J. Prost J.F. Biochemistry. 2003; 42: 15300-15309Crossref PubMed Scopus (118) Google Scholar, 11.Greenstein A.E. Grundner C. Echols N. Gay L.M. Lombana T.N. Miecskowski C.A. Pullen K.E. Sung P.Y. Alber T. J. Mol. Microbiol. Biotechnol. 2005; 9: 167-181Crossref PubMed Scopus (82) Google Scholar, 12.Narayan A. Sachdeva P. Sharma K. Saini A.K. Tyagi A.K. Singh Y. Physiol. Genomics. 2007; 29: 66-75Crossref PubMed Scopus (76) Google Scholar, 13.Kang C.M. Abbott D.W. Park S.T. Dascher C.C. Cantley L.C. Husson R.N. Genes Dev. 2005; 19: 1692-1704Crossref PubMed Scopus (317) Google Scholar, 14.Deol P. Vohra R. Saini A.K. Singh A. Chandra H. Chopra P. Das T.K. Tyagi A.K. Singh Y. J. Bacteriol. 2005; 187: 3415-3420Crossref PubMed Scopus (81) Google Scholar, 15.Cowley S. Ko M. Pick N. Chow R. Downing K.J. Gordhan B.G. Betts J.C. Mizrahi V. Smith D.A. Stokes R.W. Av-Gay Y. Mol. Microbiol. 2004; 52: 1691-1702Crossref PubMed Scopus (198) Google Scholar, 16.O'Hare H.M. Duran R. Cervenansky C. Bellinzoni M. Wehenkel A.M. Pritsch O. Obal G. Baumgartner J. Vialaret J. Johnsson K. Alzari P.M. Mol. Microbiol. 2008; 70: 1408-1423Crossref PubMed Scopus (134) Google Scholar, 17.Sharma K. Gupta M. Pathak M. Gupta N. Koul A. Sarangi S. Baweja R. Singh Y. J. Bacteriol. 2006; 188: 2936-2944Crossref PubMed Scopus (85) Google Scholar, 18.Greenstein A.E. MacGurn J.A. Baer C.E. Falick A.M. Cox J.S. Alber T. PLoS. Pathog. 2007; 3: e49Crossref PubMed Scopus (75) Google Scholar, 19.Park S.T. Kang C.M. Husson R.N. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 13105-13110Crossref PubMed Scopus (74) Google Scholar). Protein kinases A and B, encoded by pknA and pknB, respectively, are a part of the same operon that also contains the cistrons of protein phosphatase pstP, rodA (involved in cell shape control), and pbpA (involved in peptidoglycan synthesis). Overexpression of PknA in Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in a deviation from normal cell morphology with the cells forming an elongated and branched structure, whereas overexpression of PKnB in M. bovis BCG results in the formation of widened and bulging cells (13.Kang C.M. Abbott D.W. Park S.T. Dascher C.C. Cantley L.C. Husson R.N. Genes Dev. 2005; 19: 1692-1704Crossref PubMed Scopus (317) Google Scholar). Recent antisense RNA-mediated PknF knockdown studies in M. tuberculosis and results from overexpression of PknF in Mycobacterium smegmatis show effects on cell division, growth rate, morphology, and glucose transport (14.Deol P. Vohra R. Saini A.K. Singh A. Chandra H. Chopra P. Das T.K. Tyagi A.K. Singh Y. J. Bacteriol. 2005; 187: 3415-3420Crossref PubMed Scopus (81) Google Scholar). Protein kinase G (PknG) has been shown to play a role in the survival of the pathogen in host macrophages by modulating phagosome-lysosome fusion (lysosomal transfer) after macrophages phagocytose mycobacterium (20.Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (446) Google Scholar). The identification of the direct substrates of protein kinases is a key factor in determining their role in intracellular signal transduction. Several substrates of PknA and PknB have been identified. These include PbpA, a protein essential for cell division; conserved hypothetical proteins Rv1422 and Wag31; Forkhead-associated domain-containing proteins GarA and Rv0020c; mycolic acid synthesis pathway proteins KasA, KasB, and β-ketoacyl-ACP synthase III (mtFabH); the cell division protein FtsZ; MurD, a ligase involved in the process of peptidoglycan synthesis, N-acetylglucosamine-1-phosphate uridyl-transferase (GlmU), a key enzyme that synthesizes UDP-N-acetylglucosamine; and sigma factor SigH and anti-sigma factor RshA (13.Kang C.M. Abbott D.W. Park S.T. Dascher C.C. Cantley L.C. Husson R.N. Genes Dev. 2005; 19: 1692-1704Crossref PubMed Scopus (317) Google Scholar, 19.Park S.T. Kang C.M. Husson R.N. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 13105-13110Crossref PubMed Scopus (74) Google Scholar, 21.Dasgupta A. Datta P. Kundu M. Basu J. Microbiology. 2006; 152: 493-504Crossref PubMed Scopus (134) Google Scholar, 22.Villarino A. Duran R. Wehenkel A. Fernandez P. England P. Brodin P. Cole S.T. Zimny-Arndt U. Jungblut P.R. Cervenansky C. Alzari P.M. J. Mol. Biol. 2005; 350: 953-963Crossref PubMed Scopus (128) Google Scholar, 23.Grundner C. Gay L.M. Alber T. Protein Sci. 2005; 14: 1918-1921Crossref PubMed Scopus (77) Google Scholar, 24.Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 25.Veyron-Churlet R. Molle V. Taylor R.C. Brown A.K. Besra G.S. Zanella-Cleon I. Futterer K. Kremer L. J. Biol. Chem. 2008; 284: 6414-6424Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 26.Thakur M. Chakraborti P.K. J. Biol. Chem. 2006; 281: 40107-40113Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 27.Thakur M. Chakraborti P.K. Biochem. J. 2008; 415: 27-33Crossref PubMed Scopus (54) Google Scholar, 28.Parikh A. Verma S.K. Khan S. Prakash B. Nandicoori V.K. J. Mol. Biol. 2008; 386: 451-464Crossref PubMed Scopus (98) Google Scholar). PknA-mediated phosphorylation events modulate the activities of the cell division protein FtsZ, and the proteins involved in mycolic acid synthesis, mtFabH, KasA, and KasB (24.Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 25.Veyron-Churlet R. Molle V. Taylor R.C. Brown A.K. Besra G.S. Zanella-Cleon I. Futterer K. Kremer L. J. Biol. Chem. 2008; 284: 6414-6424Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 26.Thakur M. Chakraborti P.K. J. Biol. Chem. 2006; 281: 40107-40113Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). PknB-mediated phosphorylation of anti-sigma factor RshA negatively regulates its interaction with sigma factor SigH (19.Park S.T. Kang C.M. Husson R.N. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 13105-13110Crossref PubMed Scopus (74) Google Scholar). We have recently shown that PknB-mediated phosphorylation of GlmU modulates its acetyltransferase activity (28.Parikh A. Verma S.K. Khan S. Prakash B. Nandicoori V.K. J. Mol. Biol. 2008; 386: 451-464Crossref PubMed Scopus (98) Google Scholar). PknH, another transmembrane STPK, has been shown to control the embCAB operon through a regulatory protein EmbR (17.Sharma K. Gupta M. Pathak M. Gupta N. Koul A. Sarangi S. Baweja R. Singh Y. J. Bacteriol. 2006; 188: 2936-2944Crossref PubMed Scopus (85) Google Scholar). In addition, Rv0681 and DacB1 proteins have been identified as targets for PknH; however, the functional significance of these phosphorylations have not yet been worked out (29.Zheng X. Papavinasasundaram K.G. Av-Gay Y. Biochem. Biophys. Res. Commun. 2007; 355: 162-168Crossref PubMed Scopus (37) Google Scholar). PKnF has been implicated to play a role in glucose transport via regulation of an ABC transporter, Rv1747 (30.Molle V. Soulat D. Jault J.M. Grangeasse C. Cozzone A.J. Prost J.F. FEMS Microbiol. Lett. 2004; 234: 215-223Crossref PubMed Google Scholar, 31.Curry J.M. Whalan R. Hunt D.M. Gohil K. Strom M. Rickman L. Colston M.J. Smerdon S.J. Buxton R.S. Infect. Immun. 2005; 73: 4471-4477Crossref PubMed Scopus (49) Google Scholar). PknD-mediated phosphorylation of anti-anti-sigma factor Rv0516c inhibits its binding to anti-anti-sigma factor, Rv2638, suggesting a role for PknD in regulating transcription in M. tuberculosis (18.Greenstein A.E. MacGurn J.A. Baer C.E. Falick A.M. Cox J.S. Alber T. PLoS. Pathog. 2007; 3: e49Crossref PubMed Scopus (75) Google Scholar). In this study, we set out to characterize the serine/threonine protein kinase K and identify its substrates. PknK is a large protein (1100 amino acids). Although the N-terminal 290 amino acids are homologous to eukaryotic-like serine/threonine kinase domains, the C-terminal residues show homology with the regulatory region of Escherichia coli transcription regulator MalT. The PknK contains an ATP/GTP-binding site (P-loop) and a putative PDZ domain between amino acids residues 368–375 and 465–533, respectively. Here, we show that the C-terminal region is important for the activity of PknK. Results show that the transcription factor VirS is a PknK substrate and further identify four proteins of the mymA (mycobacterial monooxygenase) operon to be novel substrates of PknK. We also demonstrate that VirS is a bona fide substrate of PknK, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA by ∼2.5-fold. Luciferase reporter assays show that PknK-mediated phosphorylation regulates VirS-mediated transcription from mym promoter region, suggesting a role for PknK in regulating the expression and possibly the activity of the mymA operon proteins. Chemicals, Reagents, and Radioisotopes—The restriction and DNA-modifying enzymes pMAL-c2X vector and Amylose resin were purchased from New England Biolabs. Primers and analytical grade chemicals were purchased from Sigma-Aldrich. [γ-32P]ATP was from PerkinElmer Life Sciences and GE Healthcare. Inorganic [32P]orthophosphate (H 323PO4) was obtained from Board of Radiation and Isotope Technology (Hyderabad, India). pENTR/d-TOPO kit was purchased from Invitrogen, and pGEX-4T2 was purchased from GE Healthcare. The pC6-2 cloning vector and caspase-6 expressing clone were kind gifts from Dr. Sanjeev Galande (National Centre for Cell Science, Pune, India). Cloning, Expression, and Purification of PknK and PknK-KD—The pknK gene of M. tuberculosis was cloned by amplification of the gene (∼3.3 kb) from M. tuberculosis BAC clone Rv48 (BAC library kindly provided by Dr. Cole) using primers PknK-F1 and PknK-R1 (supplemental Table S1) and Phu DNA polymerase (NEB), followed by cloning the amplicon so obtained into pENTR/Directional-TOPO vector (Invitrogen). The kinase domain pknK-KD (∼1.2 kb) was similarly cloned using primers PknK-F1 and PknK-R2 (supplemental Table S1). The pknK and pknK-KD genes were subcloned into pMAL-c2X expression vector (NEB) using XbaI-HindIII to create the plasmids pMAL-PknK and pMAL-PknK-KD, respectively. pMAL-PknK-TATA and pMAL-PknK-K55M were generated by overlapping PCR mutagenesis using PknK-TATA F and R and PknK-K55M F and R primers (supplemental Table S1). The veracity of all clones was checked by sequencing. The pMAL expression plasmid constructs were transformed into E. coli BL21 (DE3)-CODON PLUS cells (Stratagene). Fresh transformants were grown in 500–1000 ml of LB medium containing 100 μg/ml ampicillin to a cell density of ∼A600 of 0.6. The cells were induced with 1 mm IPTG, 5The abbreviations used are: IPTG, isopropyl β-d-thiogalactopyranoside; STPK, serine/threonine protein kinase; MBP, maltose-binding protein; Mbp, myelin basic protein; GST, glutathione S-transferase; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay. and further grown for 12–16 h at 18 °C. The maltose-binding protein (MBP)-tagged proteins so overexpressed were purified following the manufacturer recommendations (NEB). Cloning, Expression, and Purification of VirS and Mym Operon Proteins—The genes encoding VirS (Rv3082), Mym (Rv3083), LipR (Rv3084), and FadD13 (Rv3089) as well as genes encoding for Rv3085, Rv3086, Rv3087, and Rv3088 were PCR-amplified from M. tuberculosis H37Rv genomic DNA (kindly provided by the Tuberculosis Vaccine Testing and Research Materials Contract, Colorado State University), using the corresponding forward and reverse primers (supplemental Table S1) and Phu DNA polymerase, followed by cloning of the amplicons into pENTR/Directional-TOPO vector (Invitrogen). The virS and mym genes were subcloned into pC6-2 expression vector (32.Purbey P.K. Jayakumar P.C. Patole M.S. Galande S. Nat. Protoc. 2006; 1: 1820-1827Crossref PubMed Scopus (13) Google Scholar) that has a caspase-6 cleavage site (VEMD) inserted into the BamHI site of pGEX-4T2 (Invitrogen), using NotI digestion. The genes lipR, Rv3085, Rv3086, Rv3088, and fadD13 were subcloned into pC6-2 using BamHI-XhoI digestion. The gene Rv3087 was subcloned into pMAL-C2X expression vector using XbaI-HindIII digestion. MBP-tagged Rv3087 was purified as described above. Caspase-6 site-containing GST-tagged proteins were overexpressed as described above. The harvested cells were then resuspended in (5 ml/1 g cells) STE buffer (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 1 mm EDTA) containing protease inhibitors. Lysozyme was added to a final concentration of 100 μg/ml, and lysate was incubated on ice for 15 min. DTT was added to a final concentration of 5 mm followed by the addition of N-laurylsarcosine to a final concentration of 1.5%. The contents were vortexed for 5 s followed by sonication to complete the lysis. Triton X-100 was added to a final concentration of 2%, and the samples were rocked at 4 °C for 30 min. The supernatant was clarified by centrifugation at 10,000 × g for 30 min. Binding to the GST beads and the elution of the GST fusion protein was essentially carried out according to the manufacturer's recommendations (GE Healthcare). Caspase-6 purification was carried out according to the protocol described (32.Purbey P.K. Jayakumar P.C. Patole M.S. Galande S. Nat. Protoc. 2006; 1: 1820-1827Crossref PubMed Scopus (13) Google Scholar). After purification, the proteins were dialyzed against storage buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 20% glycerol. In Vitro Kinase Reaction and Phosphoamino Acid Analysis—In vitro kinase assays were performed by incubating 1–10 pmol of PknK, PknK-K55M, or PknK-KD, and 5 μg of myelin basic protein (Mbp) in a 40-μl reaction volume containing 25 mm HEPES (pH 7.4), 5 mm MnCl2, 15 mm MgCl2, 1 mm DTT, 1 μm ATP, and 10 μCi of [γ-32P]ATP for 15 min at 30 °C. The reactions were stopped by the addition of 20 μl of 4× SDS sample buffer followed by boiling for 5 min. For the reactions with various putative substrates, kinase reactions were carried out with 5 pmol of PknK and 50–75 pmol of substrate in 40 μl of reaction volume containing 25 mm HEPES (pH 7.4), 20 mm magnesium acetate, 1 mm DTT, 10 μm ATP, and 10 μCi of [γ-32P]ATP. The reactions were resolved on 8–15% gradient SDS-PAGE gels and transferred to nitrocellulose membranes followed by autoradiography. Bands corresponding to the labeled protein of interest were excised from the filter and digested with trypsin. Tryptic peptides were analyzed by two-dimensional resolution on thin layer cellulose plates as described (33.Boyle W.J. van der G.P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Aliquots of the tryptic peptide mixes were further processed for phosphoamino acid analysis as described (33.Boyle W.J. van der G.P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Western Blot Analysis—The NdeI-SalI fragment of pknK was subcloned into pET22b, and thus the PknK-KD289 was overexpressed and partially purified. Polyclonal antibodies to PknK were raised in rabbit by injecting an emulsion containing Freund's incomplete adjuvant and gel-eluted PknK-KD289. For Western blot analysis, varying amounts of purified PknK-KD or M. tuberculosis whole cell extract or subcellular fractions were resolved by SDS-PAGE and transferred onto nitrocellulose membrane. After blocking the membrane with 5% nonfat dry milk in PBST (phosphate-buffered saline containing 0.1% Tween 20), the blot was incubated with rabbit anti-PknK polyclonal antibodies (1:3000 dilution) containing 2.5% bovine serum albumin and 2.5% nonfat dry milk in PBST, overnight at 4 °C. After washing, the blots were incubated with donkey anti-rabbit horseradish polyclonal antibodies (1:5000 dilution) for 1 h at room temperature in PBST containing 5% nonfat milk. Following washing, the blots were developed using West Pico ECL kit (Pierce). Purification of VirS and p-VirS—pMAL-C2X or pMAL-PknK constructs were digested with HindIII followed by end filling. These linear DNAs were digested with NdeI, and the fragment released (MBP or MBP-pknK genes) was cloned into NdeI-EcoRV sites of pDUET vector (Novagen). The gene encoding VirS was subcloned into the NotI site in the first multiple cloning site of pDUET vector. Expression constructs pDUET-MBP-VirS or pDUET-PknK-VirS were transformed into E. coli BL21 Codon Plus cells. Exponentially growing cultures (A600 of ∼0.6) were induced with 0.5 mm IPTG and further grown for 6 h at 18 °C. The cells were harvested and lysed by sonication in buffer A (20 mm Tris-HCl, pH 7.4, containing 10% glycerol, 150 mm NaCl, and 5 mm β-mercaptoethanol and protease inhibitors). The cell lysates containing His6 fusion proteins were mixed with equilibrated nickel-nitrilotriacetic acid-agarose affinity resins. After 2 h of incubation, resin was washed with 4 column volumes of buffer A containing 20 mm imidazole and 0.5 m NaCl. His-tagged VirS and p-VirS were eluted with buffer A containing 150 mm imidazole. The imidazole was dialyzed out using buffer A containing 20% glycerol. Purified proteins were estimated by Bradford assay, and the estimations were verified by resolving proteins on SDS-PAGE along with 0.3–1.5 μg of bovine serum albumin. Metabolic Labeling in E. coli—E. coli transformants harboring pDUET-MBP-VirS or pDUET-PknK-VirS were grown in 5 ml of LB medium containing 100 μg/ml ampicillin to a cell density of ∼A600 of 0.6. The cells were induced with 0.1 mm IPTG and further grown for 4 h at 18 °C. The cultures were harvested, washed with 5 ml of low phosphate medium (0.2% bacto-tryptone, 0.4% casamino acids, 0.4% glucose, 100 mm Tris-HCl, pH 7.5, 20 mm KCl, 80 mm NaCl, 20 mm NH4Cl, 0.1 mm CaCl2, and 1 mm MgSO4) and resuspended in 1 ml of low phosphate medium supplemented with 1 mCi of [32P]orthophosphate and 0.1 mm IPTG and was further grown at 18 °C for 4 h. The cells were harvested and lysed using a bead beater in buffer containing phosphate-buffered saline, 5% glycerol, 5 mm DTT, and protease inhibitors. The cell lysate was clarified, and the lysates containing His fusion protein were rocked with equilibrated nickel-nitrilotriacetic acid-agarose affinity beads for 4–6 h at 4 °C. The beads were then thoroughly washed with lysis buffer containing 20 mm imidazole and resuspended in SDS sample buffer. The digested samples were resolved on SDS-PAGE followed by transfer to nitrocellulose membrane and autoradiography. Electrophoretic Mobility Shift Assay (EMSA)—The mym promoter region of M. tuberculosis was amplified from the BAC clone Rv48 using primers MymP-F and MymP-R (supplemental Table S1) and Phu DNA polymerase (NEB), followed by cloning the amplicon so obtained into pENTR/Directional-TOPO vector (Invitrogen). This was used as the template for amplifications to obtain substrate for use in EMSAs. The substrate so obtained was purified and radiolabeled in a 40-μl reaction containing ∼5 pmol of substrate, 20 μCi of [γ-32P]ATP (specific activity 6000 Ci/mmol) and 20 units of polynucleotide kinase PNK in PNK buffer (Roche Applied Science), according to the manufacturer's recommendations. Radiolabeled PCR fragment was purified free of [γ-32P]ATP and PNK using nucleotide removal kit (Qiagen). EMSAs were carried out in a 15-μl reaction consisting of 7 ng (70 fmol; ∼50000 cpm) of labeled DNA probe and various concentrations of VirS or VirSΔ or pVirS in binding buffer (50 mm Tris-HCl, pH 6.8, 0.5 mm EDTA, 100 mm KCl, 0.5 mm DTT, 0.5 mm MgCl2, 100 ng/μl poly(dI-dC), 250 ng/μl bovine serum albumin, and 5% glycerol) for 10 min at 37 °C. The reactions were electrophoresed at 4 °C on 8% nondenaturing polyacrylamide gels (75:1-acrylamide:bis) in 1× TBE buffer for 1 h at 200 V, followed by autoradiography. Luciferase Reporter Assays—The Hsp60 promoter in the M. tuberculosis expression vector pVV16 (kindly provided by Tuberculosis Vaccine Testing and Research Materials Contract, Colorado State University) was replaced with UV15 promoter, which was amplified from pSE100 vector (a kind gift from Dr. Sabine Ehrt). The multiple cloning site of pVV16 was lengthened by cloning in an oligonucleotide containing desired restriction sites, and a second copy of the UV15 promoter was inserted after the unique NotI site (details of this new shuttle vector for expression in mycobacterium will be reported elsewhere). The mym promoter region was amplified from the BAC clone Rv48 using primers MymP-F2 and MymP-R1 (supplemental Table S1), and the amplicon obtained was cloned into pENTR/Directional-TOPO vector (Invitrogen). The luciferase reporter gene was amplified from pcDNA3-Luciferase reporter construct using primers LucF and LucR, and the amplicon was cloned into pENTR/Directional-TOPO vector (Invitrogen). The mym promoter region was cloned upstream of the luciferase gene (luc) to generate pENTR-ML. Luc or mym promoter-luc inserts were subcloned into the unique KpnI site in the modified pVV16 construct. PknK gene was amplified using primers PknK-F2 and PknK-R2 and cloned into the unique XbaI site downstream of the His6 tag. The virS gene was amplified using primers VirS-F2 and VirS-R2 and cloned into the unique HindIII site upstream of C-terminal FLAG tag and downstream of a second copy of the UV15 promoter. The various constructs made are shown in Fig. 7A. These constructs were electroporated into M. smegmatis mc2155. Fresh transformants were grown in 100 ml of Middle Brook 7H9 medium (pH 6.8) containing 25 μg/ml kanamycin, 0.05% Tween 80, and 1% ADC supplement (BD Diagnostic Systems) with aeration, to a cell density of ∼A600 of 2.0. The cultures were centrifuged and lysed using a bead beater, and the luciferase assays were performed according to the manufacturer's recommendations (Promega). The reactions were analyzed using a Berthold luminometer. Although Protein Kinase K Is Autophosphorylated Only on Threonine Residues, It Phosphorylates Mbp on Both Serine and Threonine Residues—The gene encoding protein kinase K was cloned into pMAL-c2X vector, overexpressed, and purified as described above. Based on the sequence homology with other known kinases, Lys55 of PknK was predicted to be the invariant lysine essential for catalytic activity (34.Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2296) Google Scholar). We mutated lysine 55 to methionine (K55M) and overexpressed and purified PknK-K55M. Both PknK and PknK-K55M were purified to ∼90% purity (Fig. 1A). In vitro kinase reactions with the universal substrate myelin basic protein (Fig. 1B) demonstrated that PknK phosphorylated Mbp efficiently. A band corresponding to autophosphorylated PknK could also be detected (Fig. 1B). No phosphorylation of PknK or Mbp was detectable upon mutating the lysine 55 residue (Fig. 1B), indicating this lysine residue to be essential for kinase activity, as predicted. A slight anomaly in mobility of PknK-K55M was observed (Fig. 1A), which is most likely due to the differential phosphorylation status of the two proteins. Although the active PknK has the ability to autophosphorylate itself, the inactive PknK-K55M will be unable to do so. To determine which residue(s) were autophosphorylated in PknK, phosphoamino acid analysis was carried out. The results obtained clearly revealed that only threonine residues are so phosphorylated (Fig. 1C). Similar analysis carried out with phosphorylated Mbp showed phosphorylation on both serine and threonine residues (Fig. 1D). Threonine Residues in the Activation Loop

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