Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates Linda Nova´ kova´ 1, Lenka Saskova´ 1, Petra Pallova´ 1, Jirˇı´ Janecˇek1, Jana Novotna´ 1, Alesˇ Ulrych1, Jose Echenique2, Marie-Claude Trombe3 and Pavel Branny1 1 Cell and Molecular Microbiology Division, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic 2 Departamento de Bioquı´mica Clı´nica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, Medina Allende esq Haya de la Torre, Ciudad Universitaria, Co´ rdoba, Argentina 3 Centre Hospitalo-Universitaire de Rangueil, Universite´ Paul Sabatier, Toulouse, France Keywords Searching the genome sequence of Streptococcus pneumoniae revealed the phosphoglucosamine mutase; presence of a single Ser ⁄ Thr protein kinase gene stkP linked to protein phosphoproteome; protein phosphatase; phosphatase phpP. Biochemical studies performed with recombinant StkP serine ⁄ threonine protein kinase; suggest that this protein is a functional eukaryotic-type Ser ⁄ Thr protein Streptococcus pneumoniae kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellu- Correspondence lar fractions revealed that StkP is a membrane protein. PhpP is a soluble P. Branny, Cell and Molecular Microbiology protein with manganese-dependent phosphatase activity in vitro against a Division, Institute of Microbiology, Czech synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate resi- Academy of Sciences, Vı´denˇ ska´ 1083, dues implicated in the metal binding completely abolished PhpP activity. 142 20 Prague 4, Czech Republic Autophosphorylated form of StkP was shown to be a substrate for PhpP. Fax: +420 2 41722257 These results suggest that StkP and PhpP could operate as a functional Tel: +420 2 41062658 E-mail: [email protected] pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein (Received 25 August 2004, revised 21 identification by peptide mass fingerprinting revealed two possible sub- December 2004, accepted 7 January 2005) strates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in doi:10.1111/j.1742-4658.2005.04560.x the biosynthetic pathway leading to the formation of UDP-N-acetylgluco- samine, an essential common precursor to cell envelope components. In recent years, analysis of bacterial genomes revealed Ser ⁄ Thr protein kinases (STPK) are represented by the widespread presence of eukaryotic-type Ser ⁄ Thr multigene families in Streptomyces, Mycobacterium, protein kinase as well as protein phosphatase genes in Myxococcus, and Cyanobacteria [4–7]. These bacterial many bacteria. In several cases the genes encoding groups display complex life cycle including multistage both enzymes are genetically linked and it has been cellular differentiation and the presence of multiple demonstrated that the respective gene products play protein kinase genes seems to be associated with this antagonistic roles in regulation [1–3]. Although bacter- behavior. However, the redundancy of STPKs in ial homologues of eukaryotic-type enzymes have been these microorganisms is a major hindrance in the identified and biochemically characterized, their func- study of their physiological function. It was recently tions are not well understood because of the lack of demonstrated that AfsR, Streptomyces coelicolor tran- information on their endogenous targets and activating scriptional activator, could be phosphorylated by sev- signals. eral endogenous protein kinases suggesting substrate Abbreviations GlcN-6-P, glucosamine-6-phosphate; GlcN-1-P, glucosamine-1-phosphate; GlcN-1,6-diP, glucosamine-1,6-diphosphate; GST, glutathione S-transferase; LPS, lipopolysaccharides; RNAP, RNA polymerase; STPK, serine ⁄ threonine protein kinase. FEBS Journal 272 (2005) 1243–1254 ª 2005 FEBS 1243 Ser/Thr protein kinase of S. pneumoniae L. Nova´kova´ et al. interchangeability at least in vitro [8]. In addition, the Results and Discussion phenotypes of many of the single knockouts are relat- ively weak and the function of particular protein StkP is a protein Ser/Thr kinase capable kinase cannot be clearly assigned. Streptococcus of autophosphorylation pneumoniae, with its one pair of protein kinase and phosphatase, provides a good model to study the role To characterize a putative protein kinase StkP, the of serine–threonine phosphorylation in prokaryotes. stkP gene and its truncated form containing kinase Recently, it has been demonstrated that the disruption domain were cloned in pET28b and expressed as His- of stkP gene resulted in repression of genetic transfor- tagged proteins in E. coli BL21(DE3). To rule out the mability and virulence of S. pneumoniae, suggesting an possibility that the proteins synthesized in E. coli could important role for StkP in the regulation of various be phosphorylated by an endogenous protein kinase cellular processes [9]. There are only a few examples of activity rather than by an autophosphorylation pro- such significant impact of an inactivation of single cess, the essential lysine residue of catalytic subdomain STPK on the phenotype affecting physiological func- was replaced by arginine. A stkP gene with a Lys-to- tions [3,10,11]. Arg change (pEXstkP-K42R) was also expressed in A few target substrates for bacterial STPKs have E. coli. Total cellular extracts were analyzed for auto- been identified so far. Most of them were identified phosphorylation activity in in vitro kinase assay. After due to the presence of their genes in the close vicinity incubation, the products were separated by SDS ⁄ of cognate protein kinase genes [12–14]. Another PAGE and phosphorylated proteins were identified by approach which could make the identification of sub- autoradiography. Both full-length and truncated forms strates of prokaryotic STPKs possible is a comparative of StkP were detected as phosphorylated products analysis of phosphoproteome maps of both wild-type migrating with an expected mobility (Fig. 1A, lanes 2 and corresponding mutant strains. Surprisingly, this and 3, respectively). However, about 50% decrease of approach has not been widely used. On the other hand, 32P incorporation into truncated form of StkP was in the only article reporting a comprehensive analysis observed by comparing bands intensity. Therefore, it of bacterial phosphoproteome no phosphoproteins with can be concluded that the truncated form of StkP has evident regulatory functions were detected [15]. In this altered kinetic parameters. Phosphoamino acid analysis work, we show that recombinant StkP is a functional of 32P-labeled StkP showed that full-length protein was protein kinase with Ser ⁄ Thr specificity. We also show phosphorylated by its intrinsic activity predominantly that its cognate protein phosphatase, PhpP, dephospho- at the threonine residue and weakly at the serine resi- rylates specifically autophosphorylated StkP and that due (Fig. 1C). Replacement of an essential lysine resi- its activity is strictly dependent on the presence of man- due in subdomain II involved in phosphotransfer ganese ions. In order to find out the substrate(s) of reaction resulted in a dramatic reduction of phospho- protein kinase StkP, we prepared deletion of the rylation, although the mutated protein showed residual corresponding gene in S. pneumoniae by PCR ligation 13% activity (Fig. 1B, lane 2). A similar feature was mutagenesis and allelic exchange. Cultures of the wild- observed when Pseudomonas aeruginosa protein kinase type as well as stkP null mutant strains were labeled PpkA was mutated [16]. Probably, in some particular in vivo with [33P]orthophosphate and soluble proteins cases, this mutation is insufficient to explain the com- were separated by two-dimensional gel electrophoresis. plete loss of activity and an extensive mutational ana- Mass spectrometry analysis identified six phosphory- lysis of other residues involved in phosphotransfer lated proteins. Besides the phosphoproteins which are reaction is needed. present in both the wild-type and mutant strains two As oligohistidine-tagged StkP was not capable of likely substrates of StkP were absent in mutant strain. binding to metal affinity column, a GST-chimeric We bring evidence that phosphoglucosamine mutase protein was also engineered and expressed in E. coli. GlmM, one of the putative protein kinase targets iden- Soluble fusion enzyme was purified by affinity chroma- tified, undergoes direct phosphorylation by StkP. This tography, and GST-tag was cleaved with factor Xa is the first example of an endogenous protein substrate as described in Experimental procedures. The purified modified by a serine ⁄ threonine kinase in S. pneumoniae. protein was analyzed for its cation requirements in a In addition, this is the first report in which ana- standard kinase assay with variable divalent cation con- lysis of two-dimensional phosphoproteome maps of centrations (Fig. 1D). Mn2+ cation was much more both the wild-type and STPK loss-of-function mutant effective as a cofactor than Mg2+. Maximal activation led to identification of protein kinase target in was induced in the range of 0.5–1 mm, while concentra- prokaryotes. tions between 5 and 10 mm were required for Mg2+. 1244 FEBS Journal 272 (2005) 1243–1254 ª 2005 FEBS L. Nova´kova´ et al. Ser/Thr protein kinase of S. pneumoniae A B C D E F Fig. 1. Biochemical properties