PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho‐Ser/Thr phosphatase, in <i>Mycobacterium tuberculosis</i>

Boitel, Brigitte; Ortiz-Lombardı́a, M.; Durán, Rosario; Pompeo, Frédérique; Cole, Stewart T.; Červeñanský, Carlos; Alzari, Pedro M.

doi:10.1046/j.1365-2958.2003.03657.x

Cited by 157 publications

(210 citation statements)

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“…In addition to activation loop phosphorylation, sites in the intracellular juxtamembrane region, which has been hypothesized to have a regulatory function, were identified for PknA, PknB, and PknD (19,20). Some of these sites were previously shown to be phosphorylated in vitro (19)(20)(21). In contrast to the other protein kinases, we found that PknG is phosphorylated near its amino terminus, also consistent with previous in vitro results (8).…”

Section: Resultssupporting

confidence: 90%

“…We found that four STPKs (PknA, PknB, PknD, and PknG) were phosphorylated in vivo. In addition to activation loop phosphorylation, sites in the intracellular juxtamembrane region, which has been hypothesized to have a regulatory function, were identified for PknA, PknB, and PknD (19,20). Some of these sites were previously shown to be phosphorylated in vitro (19)(20)(21).…”

Section: Resultsmentioning

confidence: 99%

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Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases

Prisic

Dankwa

Schwartz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

247

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The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.signal transduction | phosphorylation motif | phosphoproteomics A key feature of all living cells is the ability to sense environmental signals and implement adaptive changes. These inputs propagate through complex signal transduction networks whose activity is often regulated by reversible protein phosphorylation. Although Ser/Thr/Tyr protein phosphorylation-based signaling in eukaryotes has been intensively studied, the extent to which this mechanism is used in prokaryotes has only recently begun to be appreciated (1). The number of protein kinases in prokaryotes varies widely. Although many bacteria have only a few or none of these enzymes, some cyanobacteria and streptomycetes have dozens of them (2). Bacteria that do possess Ser/Thr or Tyr kinases often have complex lifestyles and depend on these kinases to regulate critical processes, such as stress adaptation, development, and virulence (2).Mycobacterium tuberculosis is an extraordinarily versatile pathogen that can exist in distinct states in the host, leading to asymptomatic latent tuberculosis (TB) infection in which bacteria are thought to be dormant, or active TB disease in which the organisms are actively replicating. To achieve these different physiologic states M. tuberculosis requires mechanisms to sense a wide range of signals from the host and to coordinately regulate multiple cellular processes. In most bacterial pathogens, the predominant phosphorylation-based signal transduction mechanism is the two-component system. The M. tuberculosis genome, however, encodes 11 Ser/Thr protein kinases (STPKs) and an equal number of two-component system sensor kinases, suggesting that these two phospho-based signaling systems are of comparable importance in this organism (3).Knowledge of the substrates of each of the M. tuberculosis STPKs is essential for understanding their function; however, only a small number of kinase-sub...

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases

Prisic

Dankwa

Schwartz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

247

304

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“…Initially, potential phospho-threonine sites on PknH to which EmbR might bind had to be inferred from data of other Mtb Pkn kinases, whereby phospho-Thr sites 171 and 173 in the activation loop of PknB had been reported to play a critical role for both kinase activation (30) and substrate binding (12). Of these sites, Thr-171 (Thr-170 in PknH) is conserved in all but 2 of the 11 Mtb Ser͞Thr kinases.…”

Section: Resultsmentioning

confidence: 99%

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Alderwick

Molle

Kremer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Ser͞Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryoticlike Ser͞Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser͞Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory Cterminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors.PknH ͉ x-ray crystallography ͉ arabinosyltransferase ͉ transcriptional regulator ͉ ethambutol

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“…We thus concluded that Thr-168 was a residue that was intermolecularly autophosphorylated. Self-activation of AfsK by phosphorylation of a single Thr residue in the activation loop is the same as for eukaryotic PKA [11,12], but a contrast with M. tuberculosis PknB [8] that self- A. AfsK, PkaG, and AfsL are from Streptomyces; Pkn kinases, from Mycobacterium; PrkC, from Bacillus; Pkn1 and Pkn4, from Myxococcus; and PKA, from Mus musculus. Highly conserved amino acids are shaded.…”

Section: Site-directed Mutagenesis Of Ser-71 Ser-128 Thr-168 and Thmentioning

confidence: 94%

“…All these kinases autophosphorylate on two or more threonine residues in the activation loop. The two phosphorylated threonines in PknB are both important for activation of its kinase activity [8,9]. On the other hand, phosphorylation of one threonine residue in the activation loop is important for mammalian cAMP-dependent protein kinase A (PKA) [11,12] and AMP-activated protein kinase (AMPK) [13].…”

mentioning

confidence: 99%

Self-activation of Serine/Threonine Kinase AfsK on Autophosphorylation at Threonine-168

et al. 2006

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A Hanks-type protein kinase AfsK autophosphorylates on threonine residue(s) and phosphorylates AfsR, a global regulator for secondary metabolism in Streptomyces coelicolor A3(2). Mass spectrometry of a tryptic digest of the autophosphorylated form of AfsKD C corresponding to the kinase catalytic domain (Met-1 to Arg-311) of AfsK, together with subsequent site-directed mutagenesis of the candidate amino acids, identified threonine-168 as a single autophosphorylation site. Threonine-168 is located in the activation loop that is known for some Ser/Thr kinases to modulate kinase activity on phosphorylation of one or more threonine residues within the loop. Consistent with this, mutant T168D, in which Thr-168 was replaced by Asp, became a constitutively active kinase; it phosphorylated AfsR to the same extent as AfsKD C produced in and purified from Escherichia coli cells during which a considerable population of it had been already phosphorylated intermolecularly. All these findings show that autophosphorylation or intermolecular phosphorylation of threonine-168 in AfsK accounts for the self-activation of its kinase activity.Keywords serine/threonine kinase, autophosphorylation, self-activation, antibiotic production, Streptomyces A number of proteins in Streptomyces, including Streptomyces coelicolor A3(2), are phosphorylated on their serine/threonine and tyrosine residues in response to developmental phases [1,2]. Recent completion of the genome projects of S. coelicolor A3(2) [3] and S. avermitilis [4] has revealed the presence of about 40 proteins having a kinase catalytic domain similar to those of the typical eukaryotic serine/threonine and tyrosine kinases. All these observations clearly show that a given Streptomyces strain possesses several protein kinases of eukaryotic type, some of which regulate growth, morphological development and secondary metabolism. Of the protein serine/threonine kinases in Streptomyces, AfsK that phosphorylates serine/threonine residues of AfsR was first discovered, representing the first instance in the bacterial world in which the ability of a bacterial Hankstype protein kinase to phosphorylate an exogenous protein has been demonstrated [5]. The AfsK-AfsR system in S. coelicolor A3(2) globally controls the biosynthesis of secondary metabolites including actinorhodin, undecylprodigiosin, methylenomycin, and a calciumdependent antibiotic [2].Autophosphorylated amino acid residues of Hanks kinases in prokaryotes, such as PrkC in Bacillus subtilis [6], PknB in Mycobacterium tuberculosis [7ϳ9], PknD, PknE and PknF in M. tuberculosis [9], and PknH in M.

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PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho‐Ser/Thr phosphatase, in Mycobacterium tuberculosis

Cited by 157 publications

References 58 publications

Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases

Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Self-activation of Serine/Threonine Kinase AfsK on Autophosphorylation at Threonine-168

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