The Escherichia coli Phosphotyrosine Proteome Relates to Core Pathways and Virulence

While phosphotyrosine modification is an established regulatory mechanism in eukaryotes, it is less well characterized in bacteria due to low prevalence. To gain insight into the extent and biological importance of tyrosine phosphorylation in Escherichia coli, we used immunoaffinity-based phosphotyrosine peptide enrichment combined with high resolution mass spectrometry analysis to comprehensively identify tyrosine phosphorylated proteins and accurately map phosphotyrosine sites. We identified a total of 512 unique phosphotyrosine sites on 342 proteins in E. coli K12 and the human pathogen enterohemorrhagic E. coli (EHEC) O157:H7, representing the largest phosphotyrosine proteome reported to date in bacteria. This large number of tyrosine phosphorylation sites allowed us to define five phosphotyrosine site motifs. Tyrosine phosphorylated proteins belong to various functional classes such as metabolism, gene expression and virulence. We demonstrate for the first time that proteins of a type III secretion system (T3SS), required for the attaching and effacing (A/E) lesion phenotype characteristic for intestinal colonization by certain EHEC strains, are tyrosine phosphorylated by bacterial kinases. Yet, A/E lesion and metabolic phenotypes were unaffected by the mutation of the two currently known tyrosine kinases, Etk and Wzc. Substantial residual tyrosine phosphorylation present in an etk wzc double mutant strongly indicated the presence of hitherto unknown tyrosine kinases in E. coli. We assess the functional importance of tyrosine phosphorylation and demonstrate that the phosphorylated tyrosine residue of the regulator SspA positively affects expression and secretion of T3SS proteins and formation of A/E lesions. Altogether, our study reveals that tyrosine phosphorylation in bacteria is more prevalent than previously recognized, and suggests the involvement of phosphotyrosine-mediated signaling in a broad range of cellular functions and virulence.     

An anti-sulfatide antibody O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the G protein α subunit in rat primary immature oligodendrocytes

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis. Of these proteins, the proteins of 59 kDa and 53/56 kDa were identified as the Src family tyrosine kinases Fyn and Lyn on the basis of their sequential immunoprecipitation with anti-Fyn and anti-Lyn antibodies, respectively. The 40 kDa protein was identified as the α subunit of the heterotrimeric G protein. These observations suggest that O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the α subunit of the heterotrimeric G protein.     

Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

Targeted disruption of the tyrosine phosphatase PTPα leads to constitutive downregulation of the kinases Src and Fyn

A role for the receptor-like protein tyrosine phosphatase α (PTPα) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPα enhances the dephosphorylation and activation of Src and Fyn [1], [2], [3]. We have generated mice lacking catalytically active PTPα to address the question of whether PTPα is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPα allele (PTPα^−/−) and lacking detectable PTPα protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPα^−/− mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPα^−/− mice. Thus, PTPα is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPα^−/− mouse brain lysates. These may be PTPα substrates or downstream signaling proteins. Taken together, the results indicate that PTPα has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.     

Interaction between O-GlcNAc Modification and Tyrosine Phosphorylation of Prohibitin: Implication for a Novel Binary Switch

Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.     

Differential hepatic protein tyrosine nitration of mouse due to aging – Effect on mitochondrial energy metabolism,quality control machinery of the endoplasmic reticulum and metabolism of drugs

Aging is the inevitable fate of life which leads to the gradual loss of functions of different organs and organelles of all living organisms. The liver is no exception. Oxidative damage to proteins and other macromolecules is widely believed to be the primary cause of aging. One form of oxidative damage is tyrosine nitration of proteins, resulting in the potential loss of their functions. In this study, the effect of age on the nitration of tyrosine in mouse liver proteins was examined. Liver proteins from young (19–22 weeks) and old (24 months) C57/BL6 male mice were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted onto nitrocellulose membranes. Proteins undergoing tyrosine nitration were identified using anti-nitrotyrosine antibody. Three different protein bands were found to contain significantly increased levels of nitrotyrosine in old mice (Wilconxon rank-sum test, p < 0.05). Electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC–MS/MS) was used to identify the proteins in these bands, which included aldehyde dehydrogenase 2, Aldehyde dehydrogenase family 1, subfamily A1, ATP synthase, H⁺ transporting, mitochondrial F1 complex, β subunit, selenium-binding protein 2, and protein disulfide-isomerase precursor. The possible impairment of their functions can lead to altered hepatic activity and have been discussed.     

Noggin proteins are multifunctional extracellular regulators of cell signaling

Noggin is an extracellular cysteine knot protein that plays a crucial role in vertebrate dorsoventral patterning. Noggin binds and inhibits the activity of bone morphogenetic proteins via a conserved N-terminal clip domain. Noncanonical orthologs of Noggin that lack a clip domain (“Noggin-like” proteins) are encoded in many arthropod genomes and are thought to have evolved into receptor tyrosine kinase ligands that promote Torso/receptor tyrosine kinase signaling rather than inhibiting bone morphogenic protein signaling. Here, we examined the molecular function of noggin/noggin-like genes (ApNL1 and ApNL2) from the arthropod pea aphid using the dorso-ventral patterning of Xenopus and the terminal patterning system of Drosophila to identify whether these proteins function as bone morphogenic protein or receptor tyrosine kinase signaling regulators. Our findings reveal that ApNL1 from the pea aphid can regulate both bone morphogenic protein and receptor tyrosine kinase signaling pathways, and unexpectedly, that the clip domain is not essential for its antagonism of bone morphogenic protein signaling. Our findings indicate that ancestral noggin/noggin-like genes were multifunctional regulators of signaling that have specialized to regulate multiple cell signaling pathways during the evolution of animals.     

Effect of 24-epibrassinolide on pea protein tyrosine phosphorylation after salinity action

The effects of the brassinosteroid hormone 24-epibrassinolide (EPB) on growth indices and tyrosine phosphorylation of soluble proteins in pea (Pisum sativum L.) leaves after salinity action were studied. Treatment of plants subjected to salinity (150 mM NaCl for 2 days) with 100 nM EPB for 2 h resulted in the resumed shoot growth. The analysis of phytohormone-induced changes in the tyrosine phosphoproteome after NaCl treatment showed that EPB suppressed tyrosine phosphorylation of proteins functioning in the dark stage of photosynthesis. This is evidently related to the recovery of photosynthetic activity and, as a consequence, of plant growth indices.     

Characterization of Phospho-(Tyrosine)-Mimetic Calmodulin Mutants

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca²⁺; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca²⁺; and iv) Tb³⁺-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.     

The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.     

Effect of EGF on transmission of the proliferative signal in infusoria Tetrahymena pyriformis

It has been established that in infusoria Tetrahymena pyriformis, transmission of a proliferative signal induced by epidermal growth factor (EGF) is not associated with autophosphorylation of receptor tyrosine kinases. In these microorganisms, EGF triggers the mitogenic pathway that involves membrane proteins of the tyrosine kinase type (without phosphorylation at tyrosine), adenylyl cyclase, and tyrosine-and Ca²⁺-dependent ERK-like kinases.     

Effect of epibrassinolide on tyrosine phosphorylation of the calvin cycle enzymes

Two-dimensional electrophoresis was used to separate proteins from crude extracts of pea (Pisum sativum L.) leaves, and thus isolated proteins were subjected to Western blot analysis with monoclonal antibodies against PY20 phosphotyrosine polypeptides. This analysis revealed 44 polypeptides phosphorylated on tyrosine residues. Phosphorylation of some of these proteins was changed under the action of epibrassinolide. Some of these polypeptides were identified by means of MALDI-TOF MS analysis. The results indicate that eight of these proteins belong to the Calvin cycle enzymes, namely, the isoforms of Rubisco large and small subunits, fructose-1,6-phosphate aldolases 1 and 2, and the precursor of α-subunit of Rubisco-binding protein. The observed changes in phosphorylation of these proteins may partly explain the effects of brassinosteroids on photosynthesis. The tyrosine phosphorylation sites were identified in silico for the fragments of polypeptides examined.     

Src-dependent Tyrosine Phosphorylation of Non-muscle Myosin Heavy Chain-IIA Restricts Listeria monocytogenes Cellular Infection

Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src.     

Phosphorylation of Enabled by the Drosophila Abelson Tyrosine Kinase Regulates the In Vivo Function and Protein-Protein Interactions of Enabled

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.     

Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.     

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.     

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria.     

Isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneumoniae

Two proteins of Klebsiella pneumoniae, termed Yor5 and Yco6, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, protein Yco6 was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive adenosine triphosphate, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, protein Yor5 was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Yor5 was able to dephosphorylate protein Yco6 previously autophosphorylated. Together, these data demonstrate that similarly to other bacterial species including Acinetobacter johnsonii and Escherichia coli, the cells of K. pneumoniae contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. Since Yco6 and Yor5 are both involved in the synthesis of capsular polysaccharide and since capsules are essential to the virulence of K. pneumoniae, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.