Centre for Biochemical Technology, Mall Road, Delhi-110 007, India1
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany2
Department of Biochemistry, University of Delhi South Campus, N. Delhi, India3
Public Health Research Institute, 455 First Avenue, NY, USA4
Department of Molecular Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany5
Author for correspondence: Yogendra Singh. Tel: +91 11 766 6156. Fax: +91 11 766 7471. e-mail: ysingh{at}cbt.res.in
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ABSTRACT |
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Keywords: tuberculosis, protein phosphorylation, glutathione S-transferase, transmembrane, mycobacteria
Abbreviations: GST, glutathione S-transferase; MBP, myelin basic protein
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INTRODUCTION |
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The regulation of eukaryotic signal transduction pathways by phosphorylationdephosphorylation of serine, threonine and tyrosine residues has been known for many years (Yarden & Ullrich, 1988 ). Recently, protein kinases have also been found to coordinate stress responses, development processes and pathogenicity in several micro-organisms (Av-Gay & Everett, 2000
). For example, Ser/Thr kinase (YpKA) is involved in the expression of virulence in Yersinia pseudotuberculosis (Galyov et al., 1993
) by disrupting the eukaryotic cytoskeleton and by reprogramming the host signalling network (Hakansson et al., 1996
).
The presence of functional Ser/Thr kinases (Peirs et al., 1997 ) and phosphorylated protein (Chow et al., 1994
) in mycobacteria was reported prior to release of the complete genome sequence of M. tuberculosis. The genomic sequence then suggested the presence of 11 putative protein kinases and 4 protein phosphatases (Cole et al., 1998
). Although the role of these mycobacterial kinases is not yet clear, they may be regulators of various metabolic processes, growth, development and interaction with host cells. Recently, inhibitors of protein kinases have been shown to prevent uptake of Mycobacterium leprae by peritoneal macrophages of mice (Prabhakaran et al., 2000
).
Recently, two putative protein Tyr phosphatases (Koul et al., 2000 ) and two functional Ser/Thr kinases, PknD (Peirs et al., 1997
) and PknB (Av-Gay et al., 1999
), from M. tuberculosis have been cloned and characterized. The present work describes the cloning, expression and characterization of PknF and PknG, two more Ser/Thr protein kinases of M. tuberculosis. The data show that pknF and pknG code for functional kinases, that PknF is a transmembrane protein, and that PknG is a cytosolic enzyme.
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METHODS |
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Plasmid construction and mutagenesis.
M. tuberculosis H37Rv genomic DNA was used as a template for amplification of two putative protein kinase genes, pknF and pknG, by PCR. The sequences of the two PCR primers for cloning pknF were 5' - GGAATTCCATGCCGCTCGCGGAAGGTTCAACGTTCC-3' for the 5' end (carrying an EcoRI site) and 5'-CCGCTCGAGTCACGGCCAGCCGTTGCTTCTGCG-3' for the 3' end (carrying an XhoI site). For cloning pknG, the sequences of the two primers were 5'-GGAATTCCATGGCCAAAGCGTCAGAGACCGAAC-3' for the 5' end (carrying an EcoRI site) and 5'-CCGCTCGAGTTAGAACGTGCTGGTGGGCCGGAC-3' for the 3' end (carrying a XhoI site). The amplified products of pknF and pknG were digested with EcoRI and XhoI, and the resulting fragments were inserted into pGEX-5X-3, which was previously digested with the same restriction enzymes. The resulting plasmids were designated pGEX-pknF and pGEX-pknG.
Site-directed mutagenesis of lysine-41 of PknF and lysine-181 of PknG to methionine was carried out as described by Kunkel et al. (1991) . The oligonucleotide used for mutagenesis of pknF was 5'-GGCCCGCAGTACCATGAGCGCGTCCTG-3', and the oligonucleotide for mutagenesis of pknG was 5'-ATGCACCAGGCCCATGAGCACCACCGG-3' (underlined bases indicate the change from lysine to methionine). The plasmids carrying the mutant genes were designated pGEX-pknF-K41M and pGEX-pknG-K181M. The pknF gene was also cloned into a eukaryotic expression vector, pCDNA3, and the resulting plasmid was designated pCDNA3-pknF. The nucleotide sequences were confirmed by sequencing using the dideoxynucleotide method (Sanger et al., 1977
).
Expression and purification of PknF and PknG.
Escherichia coli strain BL21 was separately transformed with plasmids pGEX-pknF, pGEX-pknF-K41M, pGEX-pknG and pGEX-pknG-K181M. Transformants were grown in 2YT medium (Difco) containing 100 µg ampicillin ml-1 at 37 °C until the OD600 reached 0·5. IPTG was then added to a final concentration of 0·5 mM, and cultures were grown for an additional 5 h at 37 °C with shaking. Cells were harvested by centrifugation at 5000 g for 15 min and resuspended in 20 ml sonication buffer (50 mM Tris/HCl pH 7·4, 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM PMSF and 10 µg aprotinin ml-1). The cells were then sonicated on ice for 2 min, and the sonicate was supplemented with Triton X-100 to a final concentration of 1% before centrifugation at 30000 g for 30 min at 4 °C. The supernatant fluid was incubated overnight at 4 °C with glutathione-Sepharose 4B matrix (Pharmacia Biotech). The resin bound to protein was packed into a column and washed with 5 bed vols PBS (1·44 g disodium hydrogen phosphate, 0·24 g potassium dihydrogen orthophosphate, 0·2 g potassium chloride, 8·0 g sodium chloride in 1 litre distilled water, pH 7·2). Proteins were eluted with 50 mM Tris/HCl pH 8·0 containing 1 mM DTT, 5 mM MgCl2 and 15 mM glutathione. Fractions were analysed by 10% SDS-PAGE (Laemmli, 1970 ). Fractions containing purified chimeric proteins were pooled and dialysed against PBS containing 20% glycerol and stored at -20 °C.
PknF and PknG kinase activities.
The kinase assay is based on the phosphorylation of myelin basic protein (MBP). The activities of wild-type and mutated PknF and PknG were determined by incubating 0·5 µg glutathione S-transferase (GST)-PknF, GST-PknF-K41M, GST-PknG or GST-PknG-K181M with MBP (1 µg) for 30 min at 37 °C in a kinase buffer (25 mM Tris/HCl pH 7·4 containing 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 200 µM orthovanadate and 3·7x 105 Bq [-32P]ATP). The reactions were terminated by the addition of SDS sample buffer and proteins were separated by 15% SDS-PAGE. The gels were electroblotted to nitrocellulose membranes and autoradiographed to determine the phosphorylation of MBP.
Analysis of phosphorylated residues of MBP.
Analysis of phosphoamino residues of MBP phosphorylated by PknF or PknG was carried out as described by Vincent et al. (1999) . In brief, MBP was phosphorylated by either PknF or PknG, and the protein components were separated by 15% SDS-PAGE. They were then electroblotted to a PVDF membrane. The bands corresponding to the phosphorylated MBP were excised from the membrane and acid-hydrolysed in 5·7 M HCl for 90 min at 110 °C. The acid-stable phosphoamino acids liberated by hydrolysis were separated by two-dimensional TLC and detected by autoradiography.
Production of polyclonal antiserum to PknF and PknG.
Purified GST-PknF or GST-PknG chimeric proteins (150 µg) were dissolved in 1 ml Freunds incomplete adjuvant and injected into rabbits. Subsequently, injections of 100 µg each of GST-PknF or GST-PknG in 1 ml Freunds incomplete adjuvant were given three times at 14 d intervals. Ten days after the final injection, animals were bled, and titres of anti GST-PknF were determined by ELISA as described by Harlow & Lane (1988) .
Expression of PknF in A549 cell line.
The human lung epithelial cell line A549 was maintained in Hams F12 medium (Gibco-BRL) supplemented with 10% fetal calf serum. Cells were transfected with pCDNA3 alone or the pCDNA3-pknF construct by the calcium phosphate method (Chen & Okayama, 1987 ) and lysed as described previously (Zwick et al., 1999
).
Southern blot analysis.
Analysis of the presence of pknF and pknG homologues in various species of mycobacteria was carried out as described by Reyrat et al. (1995) . In brief, genomic DNA samples (3 µg each) from M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG and M. smegmatis were digested with BamHI/NruI (for hybridization with pknF) or with SmaI (for hybridization with pknG). Digested products were separated by electrophoresis in a 1% agarose gel at 2530 V for 16 h and transferred to nitrocellulose membranes. Hybridization was performed at 66 °C using 6xSSC (1xSSC is 150 mM NaCl and 15 mM sodium citrate, pH 7·2) with a 32P-labelled pknF probe or with a 32P-labelled pknG probe. After washing with 0·1x SSC and 0·1xSDS for 20 min at 68 °C, hybrids were detected by autoradiography.
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RESULTS |
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The expressed GST-chimeric proteins (GST-PknF and GST-PknG) were purified using glutathione-Sepharose 4B matrix. The GST-PknF and GST-PknG chimeric proteins migrated in 10% SDS-PAGE as a 93 kDa protein (Fig. 1a, lane 2) and a 111 kDa protein (Fig. 1b
, lane 2), respectively. The predicted size of GST-PknF was 80 kDa (51 kDa for the PknF protein and 29 kDa for the attached GST protein). The size of the GST-PknG chimeric protein was consistent with the predicted molecular mass of the PknG protein (82 kDa) plus the attached GST protein (29 kDa). The mutated chimeric proteins, GST-PknF-K41M (Fig. 1a
, lane 3) or GST-PknG-K181M (Fig. 1b
, lane 3), were purified by the same procedure.
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PknG was detected as an 82 kDa protein predominantly in the cytoplasmic fraction of M. tuberculosis H37Rv (Fig. 4b, lane 3). PknG was also associated with cell membrane proteins (Fig. 4b
, lane 2) of M. tuberculosis H37Rv and a faint band corresponding to PknG was detected in the cell wall fraction (Fig. 4b
, lane 1). PknG expression could be detected in whole-cell lysates of M. tuberculosis H37Rv (Fig. 4b
, lane 4) and H37Ra (Fig. 4b
, lane 5), but not in whole-cell lysates from M. smegmatis (Fig. 4b
, lane 6). Thus, the two proteins differ with respect to subcellular localization.
Analysis of pknF and pknG in other mycobacterial strains
The PCR products of pknF or pknG were used as probes in Southern hybridization experiments to determine the presence of pknF or pknG homologues among different species of mycobacteria. Genomic DNA from M. smegmatis, M. bovis BCG, M. tuberculosis H37Rv and M. tuberculosis H37Ra was digested with BamHI/NruI (for hybridization with pknF) or with SmaI (for hybridization with pknG). The digested fragments were separated by agarose gel electrophoresis and blotted onto nitrocellulose membranes. Hybridizations were performed with a 32P-labelled 1350 bp pknF fragment or 32P-labelled 1747 bp pknG fragment, derived from PCR amplification and restriction digestion of the pknF (BamHI/NruI) or pknG (SmaI) genes, respectively. Hybridization results revealed that genes homologous to pknF (Fig. 5a) and pknG (Fig. 5b
) were present in members of the M. tuberculosis complex analysed in this study, but they were absent from M. smegmatis, a non-pathogenic mycobacterium. Interestingly, pknF hybridization also revealed one or two additional hybridization fragments of 2·2 kb and 5·3 kb in the M. tuberculosis H37Rv and H37Ra lanes, respectively, consistent with the finding that PknF belongs to a family of related eukaryotic-like Ser/Thr protein kinases (Cole et al., 1998
).
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DISCUSSION |
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Although phosphorylated proteins (Chow et al., 1994 ) and protein kinases (Av-Gay & Everett, 2000
; Peirs et al., 1997
) have been found in mycobacteria, their role in mycobacterial pathogenesis is poorly understood. It has been well documented that M. tuberculosis has unique properties that allow it to remain in a dormant state within the host (Parrish et al., 1998
). Protein kinases may play an important role in metabolic processes of M. tuberculosis. Therefore, we expressed and characterized two putative protein kinases, protein kinase F (PknF) and protein kinase G (PknG) of M. tuberculosis H37Rv.
PknF and PknG have a characteristic protein kinase amino acid sequence signature, including all 11 conserved domains of Ser/Thr kinases (Hanks & Quinn, 1991 ). The two enzymes phosphorylated MBP, an in vitro substrate for Ser/Thr kinases. All putative Ser/Thr kinases of mycobacteria, except for PknI, possess a lysine in the ATP-binding site (Av-Gay & Everett, 2000
). This feature is typical of Ser/Thr kinases (Av-Gay & Everett, 2000
). When lysine at position 41 (K41) of PknF or lysine at position 181 (K181) of PknG were substituted with methionine, the mutant proteins failed to phosphorylate MBP. These results suggest that PknF and PknG are functional kinases and that K41 of PknF or K181 of PknG are required for enzymic activity, consistent with their having the same catalytic properties as other Ser/Thr kinases.
Immunoblot analysis of various cellular fractions of M. tuberculosis H37Rv with anti-PknF serum revealed that PknF is localized mainly in the cell envelope. Also, predicted structural analysis of PknF from M. tuberculosis revealed that it has a transmembrane domain (Av-Gay & Everett, 2000 ). These results suggest that PknF, like PknB, is a transmembrane kinase and that it may serve as a receptor for environmental signals, as is known to be true for transmembrane kinases from Streptomyces and Myxobacteria (Nadvornik et al., 1999
). It has also been postulated that since the pknF gene is located in the ATP-binding cassette transporter operon, it may be involved in phosphate transport (Av-Gay & Everett, 2000
).
The pknF ORF encoded a translational product of 50·6 kDa, but Western blot analysis of whole-cell lysates of M. tuberculosis H37Rv and M. tuberculosis H37Ra revealed that PknF migrates during 10% SDS-PAGE as a 6770 kDa doublet. Similar results were obtained with A549 cells transfected with pCDNA3-pknF. No deleterious effects were observed with transfected A549 cells. However, this does not rule out the possible involvement of PknF in virulence. The aberrant migration of PknF during SDS-PAGE may be due to phosphorylation, as is observed in Ser/Thr kinase Pkn9 (Hanlon et al., 1997 ) and Pkn6 (Zhang, 1996
) from Myxococcus xanthus and Ser/Thr kinase PpkA from Pseudomonas aeruginosa (Motley & Lory, 1999
). The migration of PknF as a doublet protein may reflect PknF molecules having different levels of phosphorylation.
In contrast to the transmembrane kinase PknF, PknG is predominantly a cytoplasmic protein in M. tuberculosis H37Rv. A cytoplasmic localization for PknG suggests that it is a soluble protein kinase. The secreted kinase (YopO) of Yersinia and PknG have similar structures (Av-Gay & Everett, 2000 ), suggesting that PknG may have a role in the pathogenesis of mycobacteria. It has also been suggested from the genetic localization of pknG within the glutamate-binding operon that PknG may be involved in virulence by regulation of glutamate metabolism (Av-Gay & Everett, 2000
).
The genes encoding PknF and PknG, which were present in M. bovis BCG as well as in M. tuberculosis H37Rv and M. tuberculosis H37Ra, were absent from the avirulent strain M. smegmatis. This observation is consistent with our Western blot data in which we failed to detect the expression of PknG or PknF in M. smegmatis culture lysates. The absence of pknF and pknG in non-pathogenic M. smegmatis suggests possible roles in the processes specific to pathogenic mycobacteria. More experimental evidence will be necessary to demonstrate a critical function of PknF or PknG in the pathology of tuberculosis, which could open new opportunities for the development of target-specific therapies.
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ACKNOWLEDGEMENTS |
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Received 15 February 2001;
revised 23 April 2001;
accepted 25 April 2001.