Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.     

Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established

The first clearly established example of Ser/Thr/Tyr phosphorylation of a bacterial protein was isocitrate dehydrogenase. In 1979, 25 years after the discovery of protein phosphorylation in eukaryotes, this enzyme was reported to become phosphorylated on a serine residue. In subsequent years, numerous other bacterial proteins phosphorylated on Ser, Thr or Tyr were discovered and the corresponding protein kinases and P-protein phosphatases were identified. These protein modifications regulate all kinds of physiological processes. Ser/Thr/Tyr phosphorylation in bacteria therefore seems to play a similar important role as in eukaryotes. Surprisingly, many bacterial protein kinases do not exhibit any similarity to eukaryotic protein kinases, but rather resemble nucleotide-binding proteins or kinases phosphorylating diverse low-molecular-weight substrates.     

Protein phosphorylation on Ser, Thr and Tyr residues in cyanobacteria

Cyanobacteria belong to an extremely diverse group of gram-negative prokaryotes. They are all able to perform oxygen-evolving photosynthesis, but differ in morphology, ecological habitats, and physiology. This diversity is also reflected in the complexity of regulatory proteins involved in protein phosphorylation on Ser, Thr and Tyr residues. For those strains whose genomes are completely sequenced, for example, the number of genes identified so far that encode Ser/Thr and Tyr kinases range from none to 52. Genetic, molecular as well as functional genomic analyses demonstrate that Ser/Thr and Tyr kinases and phosphatases are involved in the regulation of a variety of activities according to changes in growth conditions or cell metabolism, such as cell motility, carbon and nitrogen metabolism, photosynthesis and stress response. The major challenge in the near future is to integrate these components into signaling pathways and identify their targets. Some of the Ser/Thr and Tyr kinases and phosphatases are expected to interact with classical two-component signaling pathways.     

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

Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.     

Regulation of stress-activated protein kinase signaling pathways by protein phosphatases.

Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.     

The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only a few substrate proteins and even fewer phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which two-dimensional electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than a hundred proteins. However, so far only 16 phosphorylation sites on eight of its proteins have been determined, mostly in in vitro studies. Here we performed a global, gel-free, and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites: 54 on serine, 16 on threonine, and eight on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and tricarboxylic acid cycle enzymes, several kinases, and members of the phosphoenolpyruvate-dependent phosphotransferase system. This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional analysis in bacteria.     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.     

The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.     

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome.

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.     

Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Signal integration at the level of protein kinases, protein phosphatases and their substrates.

Protein phosphorylation and dephosphorylation is one of the major mechanisms of signal integration in eukaryotic cells. It can be achieved through the phosphorylation of proteins that control the levels of second messengers, through the phosphorylation of protein kinases and phosphatases themselves, or through the reversible phosphorylation of their substrates. This article also highlights the important role of 'multi-site phosphorylation' in signal integration, in which different protein kinases produce additive, synergistic and antagonistic effects on activity by phosphorylating distinct Ser or Thr residues in a single protein.     

Regulation of vascular endothelial cell barrier function and cytoskeleton structure by protein phosphatases of the PPP family

Reversible phosphorylation of cytoskeletal and cytoskeleton-associated proteins is a significant element of endothelial barrier function regulation. Therefore, understanding the mechanisms of phosphorylation/dephosphorylation of endothelial cell cytoskeletal proteins is vital to the treatment of severe lung disorders such as high permeability pulmonary edema. In vivo, there is a controlled balance between the activities of protein kinases and phosphatases. Due to various external or internal signals, this balance may be shifted. The actual balances at a given time alter the phosphorylation level of certain proteins with appropriate physiological consequences. The latest information about the structure and regulation of different types of Ser/Thr protein phosphatases participating in the regulation of endothelial cytoskeletal organization and barrier function will be reviewed here.     

Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain.     

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

Summary: Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.     

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.