FUNCTIONAL INTERACTION BETWEEN PknA AND MstP 5.2 EXPERIMENTAL PROCEDURES

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2 FUNCTIONAL INTERACTION BETWEEN PknA AND MstP 5.1 INTRODUCTION EXPERIMENTAL PROCEDURES Generation of MstP and G117D construct Expression and purification of recombinant proteins Assessment of dephosphorylation ability Co transformation Microscopy RESULTS Effect of MstP on autophosphorylation of core Dephosphorylation ofmftsz by MstP Dephosphorylation of mmurd by MstP Role of MstP in vivo DISCUSSION

3 Cfzapter INTRODUCTION Protein phosphorylation and dephosphorylation regulate a variety of biological processes in both prokaryotes and eukaryotes. Such events in signal transduction pathways are catalyzed by protein kinases and phosphatases, respectively. In eukaryotes, proteins are phosphorylated on Ser/Thr or Tyr residues by kinases and dephosphorylated by cognate phosphatases (Hunter, 1995). In recent years, accumulation of the genome sequencing data has revealed the presence of eukaryotic-type Ser/Thr or Tyr kinases as well as phosphatases in many bacterial species (Shi et al., 1998; A v-gay and Everett 2000). Several of these kinase phosphatase pairs have been shown to play opposing roles and hence regulate diverse processes (Shi et al., 1998; Bakal and Davies, 2000). Although majority of these kinases have been studied, the studies on their regulation by phosphatases are still in their beginnings. Protein phosphatases can be classified as Ser/Thr or Tyr phosphatases. Ser/Thr phosphatases in eukaryotes are categorized into four major families known as PP 1, PP2A, PP2B and PP2C based on metal ion dependence and sensitivity to inhibitors (Cohen and Cohen, 1989). Members of PP1, PP2A and PP2B families share a common catalytic core of 220 amino acid residues and are together grouped into PPP super family (Shi et al., 1998). On the other hand, members belonging to the PP2C family are characterized by the presence of a catalytic domain of 290 amino acid residues marked by eleven conserved motifs/sub domains (Bork et al., 1996; Shi et al., 1998). These enzymes regulate a variety of signalling cascades in eukaryotes, such as stress response and Wnt pathway, splicing of trnas, sporulation and mitotic growth (Maeda et al., 1993; Robinson et al., 1994; Warmka et al., 2001). On the other hand, in bacteria, the PP2C phosphatases are involved in modulating cellular activities analogous to that found in eukaryotes. They have been shown to be involved in controlling different bacterial processes, such as spore formation, development of fruiting bodies, vegetative growth and cell segregation (Beuf et al., 1994; Irmler and Forchharnmer, 2001; Treuner-Lange et al., 2001; Adler et al., 1997). Six eukaryotic-like Ser/Thr protein phosphatases have been found to be playing role in various cellular processes in gram positive soil bacterium B. subtilis. These includes, response to environmental or nutritional stress conditions, endospore formation process and sugar transport (Obuchowski et al., 2005; Shi et al., 1998). 103

4 CMpur5 Besides these, Ser/Thr phosphatases have also been implicated in providing resistance to a variety of environmental stresses (Yang et a!., 1996). Even their role in virulence has been postulated in opportunistic pathogens, such as P. aeruginosa (Mukhopadhyay eta!., 1999). Thus, it appears that a detailed study on these proteins could be very important to have insights on their contributions in regulating kinasephosphatase mediated signalling cascades. M tuberculosis includes genes for two protein tyrosine phosphatases (PtpA, PtpB), and one protein Ser/Thr phosphatase, MstP (also known as PstP or PPP), despite the presence of eleven eukaryotic-type Ser/Thr kinases (Cole et al., 1998). While protein tyrosine phosphatases may be secreted by the pathogen to subvert the host cell signalling mechanism as shown for PtpB (Singh et al., 2003), MstP is most likely involved in reversible protein phosphorylation process in the bacterium (Chopra et al., 2003; Boitel et al., 2003). In previous reports, Ser/Thr kinase, pkna from M tuberculosis has been shown to regulate morphology of cells undergoing cell division/differentiation (Chaba et al., 2002). In mycobacterial genome, putative sequences encoding morphogenic proteins (pbpa and roda) are encompassed by pkna and mstp. Among the prokaryotic genomes sequenced to date, presence of a Ser/Thr kinase and cognate phosphatase at this location is not only a distinct feature in mycobacteria (Av-Gay and Everett, 2000), but also suggestive of regulation of morphological changes through phosphorylation/dephosphorylation cascade of events. The dephosphorylation studies revealed the ability of MstP to remove phosphate from PknA as well as the substrates phosphorylated by this kinase. However, the role of MstP on the natural substrates of PknA, as identified in previous chapters, remains to be elucidated. In this consequence, the present chapter draws out the contribution of MstP in PknA mediated phosphorylation of the endogenous substrates. Furthermore, the role of MstP in PknA mediated regulation of cell division will also be evaluated. 5.2 EXPERIMENTAL PROCEDURES Generation ofmstp and G117D construct The gene mstp was amplified from the genome of M tuberculosis H3 7Ra using primers CC39 and CC34. ~ 1.55 kb amplified product was cloned in pmal-c2, through an intermediate cloning at Smai in puc19. A mutant was generated, where glycine of motif V was replaced with aspartate (G 117D) through site directed 104

5 Cliapter 5 mutagenesis as mentioned in Fig pmal-c2 to generate MBP fusion protein. The mutant was subsequently cloned in Expression and purification of recombinant proteins MstP and the mutant G 117D were purified as MBP fusion proteins (MBP MstPIMBP-G 117D). Briefly, overnight culture of E. coli cells harboring the plasmids was reinoculated and grown at 37 C till OD 600 of 0.5 followed by induction with 0.3 mm IPTG. Cells were harvested after 3 h and lysates were prepared. The post sonicate supernatant fraction was passed through amylose resin and the MBP-MstP or MBP-G 117D were eluted using 10 mm maltose Assessment of dephosphorylation ability The dephosphorylation ability of MstP was monitored in an in vitro assay. The MBP-PknA fusion protein (1 J..Lg/15 J.!l reaction volume) either alone or in the presence of endogenous substrates (mftsz or mmurd) was mixed with 1x kinase buffer (50 mm Tris ph 7.5, 50 mm NaCl, 10 mm MnCh) and phosphorylation was initiated by adding 2 J.!Ci of [y- 32 P]ATP. Following incubation at room temperature for 20 min, MBP-MstP was added to the reaction mixtures and incubated at 37 C for additional 30 min. The reactions were stopped by adding SDS sample buffer. Samples were heated at 95 C for 5 min and resolved on 8-10 % SDS-PAGE. Gels were stained with Coomassie brilliant blue, dried in a gel dryer at 70 C for 2 h and analyzed in a phosphoimager (Molecular Imager FX, Bio-Rad, USA) using 'Quantity one' software. Subsequently, gels were also processed for autoradiography Co-transformation The co-transformation protocol was followed as mentioned in Section 2.2.4, for transformation of p19kpro-pkna and pmal-mstp in E. coli DH5a cells. Following transformation, the cells were selected on ampicillin (75 J..Lg/ml) :md hygromycin (200 J..Lg/ml). After dual selection, the clones obtained were cultured in LB broth in the presence of both the antibiotics. Although pmal vector allows the expression of protein following addition of IPTG, no induction was given due to the leaky expression of the MBP fusion proteins. The cells harboring both the plasmids were processed for microscopy. 105

6 C~pter Microscopy E. coli (strain DH5a) cells transformed/co-transformed with different plasmid constructs (p19kpro-pkna/p19kpro-k42n and pmal-mstp/pmal-g117d) were reinoculated at an initial OD 600 of 0.05 and grown further for 12 h. Cells were harvested by centrifugation and subjected to fixation with ethanol after washing with PBS. The fixed cells were collected by centrifugation and resuspended in PBS. The cells were spread on poly-l-lysine coated glass slides and examined under microscope (Carl Zeiss) following staining with 1 %methylene blue solution. 5.3 RESULTS Effect of MstP on autophosphorylation of core Figure 5.1. Dephosphorylation of PimA-core by MstP. Core was incubated with MBP-MstP for 30 min at 37 C and processed for autoradiography. Lane 1: MBP-core, lane 2: MBP-core + MBP-MstP, lane 3: MBP-core + MBP-G 117D. In Chapter 2, the mm1mum region of PknA (residues out of 431 amino acids) capable of autophosphorylation and substrate phosphorylation was identified and designated as core. The core was capable of mirroring the full length PknA in the autophosphorylation ability. Since the full length PknA was dephosphorylated by core, it was intriguing to know the role of MstP on the autophosphorylation of core. As shown in Fig. 5.1, lane 2, autophosphorylation of core diminished remarkably on incubation with MstP. In order to check the specificity of dephosphorylation, a phosphatase dead mutant was utilized. Comparison of the nucleotide derived amino acid sequence of the catalytic domain of different PP2C phosphatases revealed the presence of several signature motifs harboring conserved residues. An invariant glycine residue located in motif V was found to be present near the Mg 2 + binding site D 118 in the crystal 106

7 CMpur5 structure (Pullen et al., 2004). Mutation of G 117 with a charged residue like aspartate (G 117D) did not exhibit any phosphate hydrolyzing ability in pnpp assay. This could presumably be an outcome of introducing a drastic alteration which might have obstructed Mg 2 + binding. This mutant was, therefore chosen for monitoring the effect on dephosphorylation of core. Incubation of phosphorylated core with MBP-G 117D did not compromise the phosphorylation (Fig. 5.1, lane 3). This result indicated that in MstP, G 117 is important for its dephosphorylation ability. Furthermore, this observation justified that the decreased signal intensity of autophosphorylated PknA upon incubation with wild type MstP was not a result of interference m autophosphorylation and/or masking of the phosphorylation site of PknA due to complex formation. In fact, the effect was an outcome of the dephosphorylating ability of MstP Dephosphorylation of mftsz by MstP Available reports with MstP indicated its ability to dephosphorylate PknA as well as artificial substrates phosphorylated by this kinase (Chopra et al., 2003; Molle et al., 2006). Therefore, to evaluate the role of MstP on PknA mediated phosphorylation of the natural substrate mftsz as identified in Chapter 3, in vitro dephosphorylation assay was carried out. PknA was incubated with GST-mFtsZ for 20 min in presence of [y- 32 P]ATP followed by addition of MBP-MstP and incubation for additional 30 min at 37 C. Incubation with MstP dephosphorylated labeled PknA A B 1 2 Figure 5.2. Dephosphorylation of natural substrate mftsz by MstP. (A) PknA and mftsz were incubated with MBP-MstP for 30 min at 37 C and processed for autoradiography. Lane / : MBP-PknA, lane 2: MBP-PknA + MBP-MstP, lane 3: MBP-PknA + boiled MBP-MstP, lane 4: MBP-PknA + MBP-G117D, lane 5: GSTmFtsZ, lane 6: MBP-PknA + GST-mFtsZ, lane 7: MBP-PknA + GST-mFtsZ + MBP MstP. (B) Dephosphorylation of core and mftsz by MstP. Lane 1: MBP-core + GSTmFtsZ, lane 2: MBP-core + GST-mFtsZ + MBP-MstP. 107

8 CMpur5 as well as PknA mediated transphosphorylation of mftsz (Fig. 5.2A, lanes 2 and 7). However, dephosphorylation was not observed either in the presence of boiled MstP or G 117D (Fig. 5.2A, lanes 3 and 4). MstP was also able to dephosphorylate the transphosphorylation of mftsz by core (Fig. 5.2B, lane 2) Dephosphorylation ofltjmurd by MstP In Chapter 4, the peptidoglycan biosynthetic enzyme MurD from M tuberculosis (mmurd) was identified as one of the natural substrates of PknA. mmurd was found to be phosphorylated in presence of PknA. The ability of MstP to dephosphorylate PknA mediated transphosphorylation of mmurd was therefore examined. As evident from Fig. 5.2, lane 3, the signal corresponding to PknA as well Figure 5.3. Dephosphorylation of natural substrate mmurd by MstP. PknA and mmurd were incubated with MBP-MstP for 30 min at 37 C and processed for autoradiography. Lane I: MBP-PknA, lane 2: MBP-PknA + mmurd, lane 3: MBP-PknA + mmurd + MBP-MstP, lane 4: MBP-PknA + mmurd + MBP-G 117D. as mmurd vanished on incubation with MstP for 30 min. This indicated that MstP dephosphorylated PknA mediated transphosphorylation of mmurd. Further, no compromise in phosphorylation of PknA or mmurd could be seen on incubating the phosphorylated proteins with the phosphatase dead mutant G 117D (Fig. 5.2, lane 4). From these results it could be concluded that the Ser/Thr protein phosphatase MstP, has the ability to dephosphorylate the PknA mediated phosphorylation of the natural substrates Role of MstP in vivo The capability of MstP to dephosphorylate PknA and the natural as well as artificial substrates phosphorylated by this kinase in vitro is well established. However, no studies have been carried out to validate the PknA-MstP coupling in vivo. Therefore, it was tempting to envisage the physiological role of MstP on PknA in vivo. As mentioned in earlier report, PknA elongate the E. coli cells on its constitutive expression (Chaba et al., 2002). It was therefore hypothesized that if MstP has the ability to dephosphorylate PknA as well as the substrates phosphorylated by the kinase, and the elongation phenotype is a consequence of the phosphorylating 108

9 Cliapter 5 ability, MstP should be able to revert the phenotype generated by the kinase. In order to validate this hypothesis, mstp cloned in pmal (pmal-mstp) was transformed in E. coli harboring PknA in a Mycobacterium-E. coli shuttle vector p 19Kpro (p 19Kpro PknA). The presence of both the plasmids was confirmed by the continuous maintenance of double antibiotic present in the cultures. After dual selection on ampicillin and hygromycin, the morphology of the cells was observed by methylene blue staining. Figure 5.4. Co-expression of PknA and MstP. E. coli DH5a cells were co-transformed with different plasmid constructs and subjected to phase contrast microscopy after staining with methylene blue. Cells co-transformed with, p 19Kpro (panel a), p 19Kpro-PknA (panel b), pmal-mstp (panel c), pl9kpro-pkna and pmal-mstp (panel d). Interestingly, on coexpression of PknA and MstP, population of elongated cells was declined to a large extent and majority of cells were normal rods (Fig. 5.4, panel 'd'). This indicated that MstP was able to revert the phenotype of E. coli cells generated by PknA (Fig. 5.4, panel 'b'). However, none of the vectors alone was able to generate any morphological change (Fig. 5.4, panels ' a' and 'c'). In order to substantiate that the aforementioned phenotypic alteration mediated was an outcome of phosphorylation events and to authenticate the in vitro studies of mutants, kinase dead and phosphatase dead mutants of PknA and MstP respectively were utilized for co-transformation. Two sets of crisscross cotransformations were carried out in E. coli cells. As expected, co-transformation of kinase dead mutant of PknA (p 19Kpro-K42N) with pmal-mstp resulted in normal cells (Fig. 5.5A). Interestingly, majority of the cells transformed with p19kpro-pkna and the phosphatase mutant pmal-g 117D were elongated (Fig. 5.5B), indicating that the mutant was deficient in its dephosphorylation ability in E. coli. The results obtained with mutants confirmed that phosphorylated PknA is a substrate of MstP in vitro as well as in vivo and they are functionally coupled. 109

10 Cfzapter 5 A B Figure 5.5 Co-expression of different mutants of PimA and MstP. E. coli DHSo. cells were co-transformed with, p 19Kpro-K42N and pm AL-MstP, (A), p 19Kpro-PknA and pmal-g 117D (B). 5.4 DISCUSSION Reversible protein phosphorylation is a mechanism for signal transduction and regulation of several biological functions. These events are materialized by phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively. In bacteria, Ser/Thr protein kinases have gained enormous importance, and many of them have been shown to be regulated by their cognate phosphatases (Gaidenko et al., 2002; Rajagopal et al., 2003). The Ser/Thr phosphatases regulate various cellular processes and are, therefore, very important for bacterial survival in natural environment. The work embodied in this thesis deals with the M tuberculosis Ser/Thr kinase PknA, which in the genome is located in an operon with the only Ser/Thr protein phosphatase, MstP. In previous chapters, the autophosphorylating activity as well as transphosphorylation ability of PknA has been described. Therefore, it would be interesting to know if PknA-MstP can perform concerted action in regulating cellular processes. In earlier studies, mstp was isolated from the genome of M tuberculosis and purified as MBP fusion proteins. From the sequence comparison of PP2C family of Ser/Thr phosphatases, glycine at position 117 was identified to be a conserved residue and its mutation to aspartate abrogated its pnpp hydrolyzing ability. In the present study, both the phosphatase MstP and its mutant G 117D (conserved glycine replaced with aspartate), purified as MBP fusion proteins, were utilized for monitoring the 110

11 C~pur5 dephosphorylation ability. The dephosphorylating ability of MstP was first assessed on the phosphorylated core of PknA (as identified in Chapter 2). As expected MstP was able to dephosphorylate the phosphorylated core, however, the mutant Gl17D could not (Fig. 5.1). Earlier studies revealed that MstP can dephosphorylate PknA as well the artificial substrates such as casein phosphorylated by the kinase. However, an insight into functional linkage of the kinase and phosphatase can only be achieved if the role of phosphatase on the natural substrates of the kinase is established. In previous chapters, mftsz and mmurd were identified as natural substrates of PknA. Therefore, to gain an insight on the kinase phosphatase coupling, the effect of MstP on phosphorylated mftsz and mmurd was evaluated. Interestingly, MstP could dephosphorylate the PknA mediated phosphorylation of mftsz as well as mmurd (Figs. 5.2 and 5.3). The specificity of dephosphorylation was appraised by the inability of the phosphatase dead mutant to remove phosphate from mmurd or mftsz. In order to assess the genetic and functional linkage between PknA and MstP, p19kpro-pkna and pmal-mstp were co-transformed in E. coli strain DH5a. Since PknA on expression in E. coli resulted in elongation of cells, it was hypothesized that expression of phosphatase in such a situation could antagonize the elongation phenotype. Interestingly, expression of the phosphatase reversed the morphology of the E. coli cells generated by the expression of the kinase (Fig. 5.4). The specificity was further ensured by the co-transformation of the kinase or the phosphatase deficient mutants. While co-transformation of kinase dead mutant K42N with MstP resulted in normal cells, the transformation of phosphatases dead mutant G 117D with the active kinase PknA revealed cells with elongated morphology (Fig. 5.5B). These results substantiated the specific action ofmstp on PknA in vivo. Thus, all these lines of evidence argued that PknA and MstP can form a functional pair. The physiological relevance of PknA mediated transphosphorylation of MurD and FtsZ remains to be elucidated in future. Nonetheless, the studies presented here provide a perspective for the regulation of signal transduction, wherein PknA may phosphorylate mmurd or mftsz and the phosphorylation of these substrates is regulated through dephosphorylation by the phosphatases. However, there is no evidence that MstP per se dephosphorylated mftsz or mmurd. Based on the 111

12 C~pur5 available report it seems very likely that MstP dephosphorylated PknA, which in turn prevented the trans-phosphorylation of mftsz or mmurd. Furthermore, in vitro studies revealed the ability of MstP to dephosphorylate the phosphorylated PknA and the phenotypic effect exhibited by PknA is reversed by MstP in vivo. Taken together, both in vitro as well as in vivo studies clearly revealed that MstP antagonizes the functionality of PknA. Therefore, it is very likely that PknA-MstP work as kinase phosphatases pair, further work needs to be carried out to unravel the exact mechanism. 112