Báo cáo Y học: Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division

EVIDENCE THAT A EUKARYOTIC-TYPE SERINE/THREONINE PROTEIN KINASE FROM MYCOBACTERIUM TUBERCULOSIS REGULATES MORPHOLOGICAL CHANGES ASSOCIATED WITH CELL DIVISION

Tác giả: Rachna Chaba, Manoj Raje and Pradip K. Chakraborti

Lĩnh vực: Sinh học phân tử, Vi sinh vật học

Nội dung tài liệu: Nghiên cứu này báo cáo việc phân lập và biểu hiện của enzyme kinase serine/threonine loại eukaryotic PknA từ vi khuẩn Mycobacterium tuberculosis. PknA đã được biểu hiện dưới dạng protein dung hợp với protein gắn maltose (MBP) trong E. coli và được tinh sạch. Các thử nghiệm in vitro chỉ ra rằng PknA có khả năng tự phosphoryl hóa và phosphoryl hóa các cơ chất ngoại bào như histone và myelin basic protein. Quan trọng hơn, biểu hiện liên tục của PknA trong E. coli đã dẫn đến sự kéo dài của tế bào, cho thấy vai trò điều hòa của PknA trong quá trình phân chia tế bào. Phân tích trình tự amino acid cho thấy PknA có thể liên quan đến sự phân chia và biệt hóa tế bào.

Mục lục chi tiết:

• PRIORITY PAPER
• Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division
• Rachna Chaba, Manoj Raje and Pradip K. Chakraborti
• Institute of Microbial Technology, Chandigarh, India
• A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Rv. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of ≈ 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein-protein interaction studies revealed that PknA is capable of phosphorylating at least a ≈56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.
• Keywords: autophosphorylation; phosphorylation; PknA; serine/ threonine kinase; signal transduction.
• Signal-transduction pathways in both prokaryotes and eukaryotes often utilize protein phosphorylation as a molecular switch in regulating different cellular activities such as adaptation and differentiation. It is well known that protein kinases play a cardinal role in the process. They are grouped into two superfamilies, histidine (His) and serine/threonine (Ser/Thr) or tyrosine (Tyr) kinases, based on their sequence similarity and enzymatic specificity [1,2]. Signal transduction in prokaryotes usually uses His kinases, which autophosphorylate at histidine residues [2]. In eukaryotes, such signalling pathways are mediated by Ser/Thr or Tyr kinases, which autophosphorylate at serine/threonine or tyrosine residues [1].
• Interestingly, analysis of genome sequences revealed the presence of putative genes encoding eukaryotic-type Ser/Thr kinases in many bacterial species. A search of the Escherichia coli genome also indicated the presence of sequences exhibiting homology with eukaryotic-type Ser/Thr kinases, but they have not been characterized biochemically or functionally. Involvement of such kinases in regulating growth and development has largely been documented in soil bacteria such as Myxococcus [3-6], Anabaena [7] and Streptomyces [8,9]. In Yersinia pseudotuberculosis, YpkA has been identified as the first secretory prokaryotic Ser/Thr kinase involved in pathogenicity [10]. Besides these, eukaryotic-type Ser/Thr kinases have been implicated in virulence in opportunistic pathogens such as Pseudomonas aeruginosa [11]. Thus a detailed study of these kinases, especially in pathogenic bacteria, could produce important insights into their contributions to signal transduction. This may help in the design of drug intervention strategies in a situation where the emergence of drug-resistant strains of several pathogenic bacteria has resulted in the rapid resurgence of diseases thought to be near irradication. We focused on tuberculosis, a disease caused by Mycobacterium tuberculosis, which is responsible for considerable human morbidity and mortality world wide [12].
• In the M. tuberculosis genome, 11 putative eukaryotic-type kinases have been reported [13]. Among these Ser/Thr kinases, four (PknB, PknD, PknF, PknG) have been biochemically characterized [14-16], but their biological functions are not known. The M. tuberculosis genome sequence further indicated that the gene for a putative Ser/Thr kinase, pknA, is located adjacent to those encoding bacterial morphogenic proteins. Interestingly, the presence of a Ser/Thr kinase at this location in the mycobacterial genome is unique among prokaryotes [17]. We therefore concentrated on PknA. In this paper, we report the cloning and expression of PknA as a fusion with maltose-binding protein (MBP). Characterization of the fusion protein revealed that it is capable of phosphorylating itself as well as basic protein substrates not present in M. tuberculosis. Furthermore, we present strong evidence that the constitutive expression of this kinase causes elongation of cells in E. coli, supporting a regulatory role for PknA in cell division.
• Correspondence to P. K. Chakraborti, Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.
• Fax: + 91 172 690 585, Tel.: + 91 172 695 215,
• E-mail: pradip@imtech.res.in
• Abbreviations IPTG, isopropyl thio-β-D-galactoside;
• MBP, maltose-binding protein.
• (Received 16 November 2001, revised 3 January 2002, accepted 9 January 2002)
• MATERIALS AND METHODS
• Bacterial strains and vectors
• M. tuberculosis strain H37Ra [18] used in this study was grown at 37 °C using oleic acid/albumin/dextrose/catalase/Tween-80/glycerol-supplemented Middle brook 7H9 broth or 7H10 agar. E. coli strains DH5a and TB1 were cultured on Luria-Bertani agar or broth. Vectors such as pUC19 and PMAL-c2X were obtained from commercial sources. The Mycobacterium-E. coli shuttle vector, p19Kpro, was a gift from D. B. Young and M. Blokpoel, Imperial College School of Medicine at St Mary’s, London, UK.
• PCR amplification, site-directed mutagenesis, and construction of recombinant plasmids
• Genomic DNA was isolated from M. tuberculosis strain H37Ra as described elsewhere [19] except that the spheroplast lysis step was carried out for 24 h at 37 °C with SDS (4%) and proteinase K (500 µg·mL⁻¹). DNA thus obtained was used for PCR amplification of pknA. The Expand Long Template PCR system (mixture of Pwo and Taq DNA polymerases; Roche Molecular Biochemicals) was used for this purpose. The forward (CC7: 5′-CATATGAGCCCCCGAGTTGG-3′) and reverse (CC8: 5′-TCATTGCGCTATCTCGTATCGG-3′) primers were designed on the basis of the published M. tuberculosis genome sequence [13] of pknA (Rv0015c). Oligonucleotides used in this study were custom-synthesized from IDT, Coralville, IN, USA. PCR was carried out for 30 cycles (denaturation, 95 °C for 30 s per cycle; annealing, 50 °C for 30 s per cycle; elongation, 68 °C for 2 min for first 10 cycles and then for the remaining 20 cycles the elongation step was extended for an additional 20 s in each cycle).
• PCR was also used to generate the K42N (replacement of lysine by asparagine at residue 42) point mutant of PknA. Two forward primers, CC58 (5′-CACAGGAATTCCATATGAGCCCCCGAGTTGG-3′), CC62 (5′-GTGTTGCGG TGAATGTGCTCAAGAGCG-3′) and two reverse primers, CC61 (5′-CTGCCCGGTGGGGGTGATCAAGATG-3′), CC63 (5′-CGCTCTTGAGCACATTCACCGCAACAC-3′), were synthesized. Base mismatches (underlined bases) for the desired mutations were incorporated in primers CC62 and CC63. To generate the mutant, two sets of primary and one set of secondary PCR reactions were carried out as described elsewhere [20] using the gel-purified pknA (≈ 1.3 kb) as template. Primary reactions were carried out with primers CC58/CC63 and CC61/CC62, while for secondary reactions, PCR primers CC58 and CC61 were used. Thus, the K42N mutation was contained within the amplified ≈ 460-bp fragment of pknA, which has a unique Xhol site in addition to the EcoRI and Ndel sites incorporated in the primer CC58.
• All manipulations with DNA were performed by standard methods [21]. Restriction/modifying enzymes and other molecular biological reagents used in this study were obtained from New England Biolabs. After PCR amplification, pknA was treated with Klenow, and the blunt-ended fragment was cloned at the Smal site of pUC19 (pPknA). Plasmid DNA was prepared after transformation of pPknA in E. coli strain DH5a and sequenced in an automated sequencer (ABI; PE Applied Biosystems).
• To monitor expression of PknA fused with MBP, E. coli vector pMAL-c2X was used. After digestion of pPknA and PMAL-c2X with NdeI and BamHI, respectively, they were treated with Klenow to obtain blunt-ended fragments. Both these fragments were further restriction-digested with HindIII, ligated and transformed in E. coli strain TB1 to obtain clones containing the plasmid (pMAL-PknA) bearing in-frame fusion of ≈ 1.3 kb pknA (confirmed by junction sequencing) at the 3′ end of MBP. To express the K42N mutant as an MBP fusion protein, a ≈ 460-bp fragment of mutated pknA was digested with EcoRI/Xhol and substituted for the corresponding wild-type fragment in the PMAL-PknA backbone. The resulting construct, pMAL-K42N, was sequenced to confirm the mutation.
• pknA or the K42N mutant was also cloned in the Mycobacterium-E. coli shuttle vector p19Kpro [22] to obtain the constitutive expression plasmids (p19Kpro-PknA or p19Kpro-K42N). The strategy adopted was same as for construction of pMAL-PknA. To clone pknA in an antisense orientation, pPknA was initially digested with Ndel and treated with Klenow to obtain a blunt-ended fragment. After restriction digestion with BamHI, this fragment was subsequently ligated to p19Kpro, which was already digested with BamHI and EcoRV. The antisense construct of pknA was designated p19Kpro-aPknA. All three constructs, p19Kpro-PknA, p19Kpro-K42N and p19Kpro-aPknA were transformed in E. coli strain DH5a. Clones carrying the gene of interest were confirmed at all steps by restriction analysis and Southern-blot hybridization. The probe (PCR-amplified pknA) used was radiolabelled by random priming with [α-32P]CTP (BRIT, Hyderabad, India).
• Expression of recombinant protein
• PMAL-PknA or pMAL-K42N cultures were grown at 37 °C and induced with 0.3 mm isopropyl thio-β-D-galactoside (IPTG) at an A600 of 0.5. Cells were harvested after 3 h, lysates were prepared, and expression was monitored by SDS/PAGE (8% gel) and Coomassie Brilliant Blue staining. To find out the solubility of the expressed fusion protein, after induction cells were suspended in lysis buffer and sonicated. Supernatant and pellet fractions obtained after sonication were subjected to SDS/PAGE. Finally, the fusion protein was purified by affinity chromatography on an amylose column according to the manufacturer’s instructions (New England Biolabs). In a similar manner, MBP-βgal fusion protein expressed by pMAL-c2X was also purified for its use as a control. To examine the constitutive expression of the protein and its solubility, overnight cultures (at 37 °C) of constructs in p19Kpro were processed in the same way as pMAL-PknA except that IPTG induction was not required.
• Kinase assay
• The ability of PknA or the K42N mutant, as a purified fusion protein with MBP, to autophosphorylate and phosphorylate exogenous substrates such as histone (from calf thymus, type II-AS; Sigma) or myelin basic protein (from bovine brain; Sigma) was determined in an in vitro kinase assay. Aliquots (usually 800 ng to 6 µg in 20 µL reaction volume) of fusion protein (MBP-PknA or MBP-K42N or MBP-βgal) were mixed with 1× kinase buffer (50 мм Tris/HCl, pH 7.5, 50 мм NaCl, 10 мм MnCl2), and the reaction was initiated by adding 2 µCi [γ-32P]ATP. After incubation at 24 °C for 20 min, the reaction was stopped by adding SDS sample buffer (30 mm Tris/HCl, pH 6.8, 5% glycerol, 2.5% 2-mercaptoethanol, 1% SDS and 0.01% bromophenol blue). Samples were boiled for 5 min and resolved by SDS/PAGE (8–12.5% gels). Gels were stained with Coomassie Brilliant Blue, dried in a gel dryer (Bio-Rad) at 70 °C for 2 h and finally exposed to Kodak X-Omat/AR film. To monitor the effect of bivalent cations, the 10 mm MnCl2 in the 1 × kinase buffer was substituted with 1, 10 or 100 mm Mn2+/Mg2+/Ca2+. The autophosphorylating ability of the constitutively expressed PknA was determined using p19Kpro-PknA-transformed E. coli extract in a similar manner.
• To identify proteins that interacted with PknA, MBP-PknA (100 µg) was immobilized on amylose resin and incubated in the presence of soluble protein extracts (250 µg) prepared from E. coli strain DH5a for 10 h at 4 °C. Amylose beads were washed (4500 g for 5 min) four times to remove unbound proteins. After suspension of washed beads in TEN buffer (20 mm Tris/HCl, pH 7.5, 200 mm NaCl and 1 mm EDTA), aliquots (12 µL) were used for phosphorylation assays.
• Western blotting
• Phosphoamino-acid analysis was carried out by Western blotting. Purified fusion proteins or cell extracts (800 ng to 3 µg protein per slot) were resolved by SDS/PAGE (8% gel) and transferred at 250 mA for 45 min to nitrocellulose membrane (0.45 µm) in a mini-transblot apparatus (Bio-Rad) using Tris/glycine/SDS buffer (48 mm Tris, 39 мм glycine, 0.037% SDS and 20% methanol, pH ≈ 8.3). Primary antibodies (anti-MBP, anti-phosphoserine, anti-phosphothreonine and anti-phosphotyrosine) used for different immunoblots were commercially available (New England Biolabs, Santa Cruz Biotech and Sigma). Horse-radish peroxidase-conjugated anti-(mouse IgG) Ig or anti-(rabbit IgG) Ig secondary antibody (Roche Molecular Biochemicals) was chosen depending on the primary antibody used, and the blots were processed by the ECL detection system (Amersham Pharmacia Biotech) following the manufacturer’s recommended protocol.
• Northern blotting
• Total RNA was isolated from cultures harbouring p19Kpro or p19Kpro-PknA plasmid by the hot phenol extraction method [23]. For Northern-blot analysis, RNA samples were electrophoresed on 1.2% agarose gel containing formaldehyde and transferred to a nylon membrane. The membrane was UV cross-linked and then hybridized with [α-32P]CTP-labelled pknA as a probe following the standard protocol [21].
• Scanning electron microscopy
• Overnight cultures (E. coli strain DH5a transformed with p19Kpro, p19Kpro-PknA, p19Kpro-aPknA or p19Kpro-K42N) were reinoculated such that initial A600 was 0.05 and grown for a further 12 h. After harvesting, cells were washed three times with ice-cold NaCl/