GCR2 is a new member of the eukaryotic lanthionine synthetase component C-like protein family

GCR2 was recently proposed to represent a G-protein-coupled receptor (GPCR) for the plant hormone, abscisic acid (ABA). We and others provided evidence that GCR2 is unlikely to be a bona fide GPCR because it is not clearly predicted to contain seven transmembrane domains, a structural hallmark for classical GPCRs. Instead, GCR2 shows significant sequence similarity to homologs of bacterial lanthionine synthetase component C (LanC). Here, we provide additional analysis of GCR2 and LanC-like (LANCL) proteins in plants, and propose that GCR2 is a new member of the eukaryotic LANCL protein family.Key words: GCR2, G-protein-coupled receptor, abscisic acid (ABA), lanthionine synthetaseSeven transmembrane (7TM) G-protein-coupled receptors (GPCRs) comprise the largest protein family in mammals, and are the most pharmacologically important receptor family, being the target of approximately half of all modern medicinal drugs. All canonical GPCRs are integral membrane proteins and are predicted to contain 7TM-spanning domains as their structural hallmark, a pattern confirmed by the high-resolution crystal structure of human β2-adrenergic GPCR.^1,2 GPCRs sense extracellular molecules and activate intracellular cell signaling via coupling with heterotrimeric G-proteins. Heterotrimeric G-protein subunits are conserved in plants, but the repertoire of heterotrimeric G-protein complexes to which they contribute in plants is much simpler than in mammals.^3,4 Liu et al. (2007) proposed that GCR2 is a GPCR for the plant hormone abscisic acid (ABA) in Arabidopsis.⁵ However, GCR2 was predicted not to be a 7TM protein when its amino acid sequence was analyzed in robust transmembrane prediction systems.^6,7 On the other hand, GCR2 has significant sequence similarity to homologs of bacterial lanthionine synthetase component C (LanC) that are found in diverse eukaryotes and which have predicted structural similarity to prokaryotic LanC.^6,7 These findings raise the possibility that GCR2 belongs to the LanC protein superfamily, rather than the GPCR superfamily.     

Peptide microarrays for high-throughput studies of Ser/Thr phosphatases

Protein phosphorylation and dephosphorylation play an important role in regulation of intracellular signal transduction pathways in the biological system. A key step in the biological characterization of phosphatases and their use as drug targets is the identification of their cellular partners and suitable substrates for potential inhibitor development. Herein we describe a microarray-based protocol to map the substrate specificity of protein Ser/Thr phosphatases. This protocol uses Pro-Q dye to sensitively and quantitatively detect the amount of dephosphorylation that occurs from many putative peptide substrates in parallel, and therefore could be used to generate the so-called peptide substrate fingerprints as well as detailed kinetic information of a target phosphatase. Excluding the synthesis of the peptide substrates, the whole protocol takes a total of 11 h to complete and in future can be readily extended to the study of other classes of phosphatases, i.e., protein tyrosine phosphatases.     

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.     

Protein Ser/Thr/Tyr Phosphorylation in the Archaea

The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms, many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology, the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial genomic, proteomic, and biochemical analyses have revealed the presence of “eukaryotic” protein kinases and phosphatases and an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights into this important regulatory mechanism.     

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.     

Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues.

MEK is a family of dual specific protein kinases which activate the extracellular signal-regulated kinases by phosphorylation of threonine and tyrosine residues. MEK itself is activated via serine phosphorylation by upstream activator kinases, including c-raf, mos and MEK kinase. Here, we report the activation phosphorylation sites of human MEK1 and yeast STE7 kinase as determined by a combination of biochemical and genetic approaches. In human MEK1, substitution of either serine residue 218 or 222 with alanine completely abolished its activation by epidermal growth factor-stimulated Swiss 3T3 cell lysates or immunoprecipitated c-raf, suggesting that both serine residues are required for MEK1 activation. Phosphopeptide analysis demonstrated that serine residues 218 and 222 of human MEK1 are the primary sites for phosphorylation by c-raf. These two serine residues are highly conserved in all members of the MEK family, including the yeast STE7 gene product, a MEK homolog in the yeast mating pheromone response pathway. Mutation of the corresponding residues in STE7 completely abolished the biological functions of this gene. These data demonstrate that MEK is activated by phosphorylation of two adjacent serine/threonine residues and this activation mechanism is conserved in the MEK family kinases.     

First structural glimpse at a bacterial Ser/Thr protein phosphatase

In this issue of Structure, we describe the crystal structure of the Ser/Thr protein phosphatase PstP from Mycobacterium tuberculosis, opening new perspectives to understand the putative roles of "eukaryotic-like" signaling elements in bacteria.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Mycobacterium tuberculosis S-adenosyl-l-homocysteine hydrolase is negatively regulated by Ser/Thr phosphorylation

S-Adenosylhomocysteine hydrolase (SahH) is known as an ubiquitous player in methylation-based process that maintains the intracellular S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) equilibrium. Given its crucial role in central metabolism in both eukaryotes and prokaryotes, it is assumed that SahH must be regulated, albeit little is known regarding molecular mechanisms governing its activity. We report here that SahH from Mycobacterium tuberculosis can be phosphorylated by mycobacterial Ser/Thr protein kinases and that phosphorylation negatively affects its enzymatic activity. Mass spectrometric analyses and site-directed mutagenesis identified Thr2 and Thr221 as the two phosphoacceptors. SahH_T2D, SahH_T221D and SahH_T2D/T221D, designed to mimic constitutive phosphorylation, exhibited markedly decreased activity compared to the wild-type enzyme. Both residues are fully conserved in other mycobacterial SahH orthologues, suggesting that SahH phosphorylation on Thr2 and Thr221 may represent a novel and presumably more general mechanism of regulation of the SAH/SAM balance in mycobacteria.     

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the α isoform of human PP2C (PP2Cα), which folds into an α/β domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn²⁺-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.     

Recent progress on the structure of Ser/Thr protein phosphatases

PP1, PP2A and PP2B, belonging to the PPP family of Ser/Thr protein phosphatases, participate in regulating many important physiological processes, such as cell cycle control, regulation of cell growth and division regulation, etc. The sequence homology between them is relatively high, and ter- tiary structure is conserved. Because of the complexity of the structure of PP2A and the diversity of its regulatory subunits, its structure is less well known than those of PP1 and PP2B. The PP2A holoen- zyme consists of a heterodimeric core enzyme, comprising a scaffolding subunit and a catalytic sub- unit, as well as a variable regulatory subunit. In this study, the subunit compositions, similarities and differences between the Ser/Thr protein phsphatases structures are summarized.     

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。     

Alphabet, a Ser/Thr phosphatase of the protein phosphatase 2C family, negatively regulates RAS/MAPK signaling in Drosophila

Signal transduction through the RAS/mitogen-activated protein kinase (MAPK) pathway depends on a diverse collection of proteins regulating positively and negatively signaling flow. We previously conducted a genetic screen in Drosophila to identify novel components of this signaling pathway. Here, we present the identification and characterization of a new gene, alphabet (alph), whose activity negatively regulates RAS/MAPK-dependent developmental processes in Drosophila and this, at a step downstream or in parallel to RAS. alph encodes a protein phosphatase 2C (PP2C) family member closely related to the mammalian PP2C alpha and beta isoforms. Interestingly, although alph gene product does not appear to be essential for viability, its elimination leads to weak but significant developmental defects reminiscent of an overactivated RAS/MAPK pathway. Consistent with this interpretation, strong genetic interactions are observed between alph alleles and mutations in bona fide components of the pathway. Together, this work identifies a PP2C of the alpha/beta subfamily as a novel negative regulator of the RAS/MAPK pathway and suggests that these evolutionarily conserved enzymes play a similar role in other metazoans. Finally, despite the relatively large size of the PP2C gene family in metazoans, this study represents only the second genetic characterization of a PP2C in these organisms.     

Noncompetitive inhibition of plant protein Ser/Thr phosphatase PP7 by phosphate

Changes in the cytoplasmic inorganic phosphate (P(i)) concentrations are an important cue for the plant cells to regulate their metabolism and phosphate homeostasis. However, phosphate sensors/receptors involved in this regulation are largely unknown. P(i) is a common nonspecific competitive inhibitor of phosphatases, usually in millimolar range. Here we report a procedure to refold recombinant Arabidopsis thaliana protein Ser/Thr phosphatase PP7 and demonstrate that PP7 is inhibited by submillimolar P(i) concentrations (IC(50) = 0.66 +/- 0.14 mM) via a mainly noncompetitive mechanism. The results indicate that PP7 may possess a specific P(i)-binding site responsible for its allosteric regulation, and suggest a possible phosphate sensor function for this protein phosphatase.     

Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.     

Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase

Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highly conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the active sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, and Lys153 in the active site of UDP-galactose 4-epimerase are located in similar positions as the corresponding Thr134, Tyr160, and Lys164, in the active site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all mutants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was used to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mutation of Tyr160 to Phe and only partly rate limiting in the reaction catalyzed by K164M and T134A dehydratases. The pH dependence of k(cat), the steady-state NADH level, and the fraction of NADH formed with saturating dTDP-xylose show shifts in the pK(a) assigned to Tyr160 to more basic values by mutation of Lys164 and Thr134. The pK(a) of Tyr160, as determined by the pH dependence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are believed to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galactose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [Liu, Y., et al. (1997) Biochemistry 35, 10675--10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a proton from the sugar to Tyr160.