Crystal Structure of the Catalytic Domain of the PknB Serine/Threonine Kinase from Mycobacterium tuberculosis

Ortiz-Lombardı́a, M.; Pompeo, Frédérique; Boitel, Brigitte; Alzari, Pedro M.

doi:10.1074/jbc.m300660200

Cited by 149 publications

(193 citation statements)

References 37 publications

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“…M. tuberculosis PknB kinase domain (1o6y) (28) and phosphorylase kinase (Phk)-peptide substrate complex (2phk) (29) crystal structures were aligned using PyMOL (DeLano Scientific) to add missing PknB-peptide contact residues in the activation loop. Activation loop residues from Phk and its substrate were changed to the corresponding PknB and peptide substrate residues so that this model should represent the conformation of the active form of PknB.…”

Section: Methodsmentioning

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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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: Methodsmentioning

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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“…However, these eukaryotic-like protein kinases lack some of the essential motifs of ePKs. Other studies have indicated that some of the eukaryotic-like protein kinases had distinct evolutionary histories, which might be even more ancient than ePKs (9,25).…”

Section: Discussionmentioning

confidence: 99%

Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases

Lai

Safaei

Pelech

2016

Journal of Biological Chemistry

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The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.Protein kinases play a pivotal role in communicating intracellular signals in eukaryotes. The family of eukaryotic protein kinases (ePKs) 3 comprises at least 568 human members, which accounts for more than 2% of the protein coding genes of the entire human genome (1). These kinases are highly conserved both in their primary amino acid sequences (2) and in the threedimensional structures (3) of their catalytic domains. Because of the central regulatory roles and the high conservation of the ePKs, the ancestry of these enzymes has become an important question in the study of the evolution of eukaryotic organisms.The majority of the kinases among the ePKs are responsible for the phosphorylation of proteins on serine or threonine residues, whereas a smaller group of protein kinases catalyzes their tyrosine phosphorylation. This branch of protein-tyrosine kinases (PTKs) arose from protein-serine/threonine kinases (STKs), which is believed to be an important event in early metazoan evolution (4, 5). Of all the STKs, there is another lumped group of diverse kinases that are described as atypical protein kinases. With little sequence identity and structural similarity to typical protein kinases, these atypical protein kinases are suggested to have diverged early in evolution and have distinct evolutionary histories (6, 7). Despite the atypical protein kinases and recently derived PTKs, the rest of the typical protein kinases constitutes a major lineage in protein kinase evolution.Eukaryotic life is believed to have evolved between 1.7 and 2.7 billion years ago, and no living representatives of the earliest eukaryotes survive today. Consequently, the actual origin of protein kinases is difficult to establish with a high degree of confidence. Firstly, protein sequences are highly degenerate, which makes the detection of sequence similarities difficult even at the superfamily level (8). Secondly, the ePKs comprise a group of very broadly expanded proteins. Loss and expansion of kin...

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“…Despite sequence identity less than 27%, prokaryotic and eukaryotic serine/threonine kinases show close conformational similarity [7,11,17]. The conformational changes in activation of these kinases with a characteristic two-lobed structure are linked to changes at regulatory sites that include phospho-acceptors in the activation loop (Fig.…”

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

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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Crystal Structure of the Catalytic Domain of the PknB Serine/Threonine Kinase from Mycobacterium tuberculosis

Cited by 149 publications

References 37 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

Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases

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

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