Mutational analysis of the Lactococcus lactis NIZO B40 exopolysaccharide (EPS) gene cluster: EPS biosynthesis correlates with unphosphorylated EpsB

AIMS: To determine the role of the EpsA, EpsB, and EpsC proteins encoded at the 5'-end of the exopolysaccharide (EPS) gene cluster in regulation of EPS production in Lactococcus lactis. METHODS AND RESULTS: Deletion and paralog-replacement mutants of epsABCD were used to determine the function of EpsA, EpsB and EpsC in EPS production and polymer chain length determination in L. lactis. EpsA and EpsB appeared to be essential for EPS biosynthesis in L. lactis, while deletion of the phosphatase (EpsC) only had a minor effect on the EPS production level. Determination of the phosphorylation state of EpsB and analysis of a C-terminally truncated EpsB variant indicate that EPS biosynthesis in L. lactis is driven by a nonphosphorylated form of EpsB. CONCLUSIONS: The data presented here show that in L. lactis, EPS production is under control of a phosphoregulatory system and that EPS biosynthesis correlates with an unphosphorylated EpsB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides molecular understanding of polysaccharide production in L. lactis that could eventually enable novel approaches to control EPS production by lactic acid bacteria during industrial fermentation processes.     

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.     

Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae

The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.     

Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae.

Vibrio cholerae secretes a number of proteins important for virulence, including cholera toxin. This process requires the products of the eps genes which have homologues in genera such as Aeromonas, Klebsiella and Pseudomonas and are thought to form a membrane-associated multiprotein complex. Here we show that the putative nucleotide-binding protein EpsE is associated with and stabilized by the cytoplasmic membrane via interaction with EpsL. Analysis of fusion proteins between EpsE and the homologous ExeE from Aeromonas hydrophila demonstrates that the N-terminus of EpsE contains the EpsL binding domain and determines species specificity. An intact Walker A box, commonly found in ATP-binding proteins, is required for activity of EpsE in vivo and for autophosphorylation of purified EpsE in vitro. These results indicate that both the kinase activity of EpsE as well as its ability to interact with the putative cytoplasmic membrane protein EpsL are required for translocation of toxin across the outer membrane in Vibrio cholerae.     

The Streptococcus pyogenes orphan protein tyrosine phosphatase,SP‐PTP,possesses dual specificity and essential virulence regulatory functions

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP‐PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP‐PTP is, therefore, questionable. Here, we demonstrate that SP‐PTP possesses dual phosphatase specificity for Tyr‐ and Ser/Thr‐phosphorylated GAS proteins, such as Ser/Thr kinase (SP‐STK) and the SP‐STK‐phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP‐PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP‐PTP displayed increased Ser‐/Thr‐/Tyr‐phosphorylation. SP‐PTP also differentially regulates the expression of ～50% of the total GAS genes, including several virulence genes potentially through the two‐component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP‐PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr‐phosphorylation in GAS and the relevance of SP‐PTP as an important therapeutic target.     

The EpsE flagellar clutch is bifunctional and synergizes with EPS biosynthesis to promote Bacillus subtilis biofilm formation

Many bacteria inhibit motility concomitant with the synthesis of an extracellular polysaccharide matrix and the formation of biofilm aggregates. In Bacillus subtilis biofilms, motility is inhibited by EpsE, which acts as a clutch on the flagella rotor to inhibit motility, and which is encoded within the 15 gene eps operon required for EPS production. EpsE shows sequence similarity to the glycosyltransferase family of enzymes, and we demonstrate that the conserved active site motif is required for EPS biosynthesis. We also screen for residues specifically required for either clutch or enzymatic activity and demonstrate that the two functions are genetically separable. Finally, we show that, whereas EPS synthesis activity is dominant for biofilm formation, both functions of EpsE synergize to stabilize cell aggregates and relieve selective pressure to abolish motility by genetic mutation. Thus, the transition from motility to biofilm formation may be governed by a single bifunctional enzyme.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.     

Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.     

Interactions of the NPXY microdomains of the low density lipoprotein receptor‐related protein 1

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY₄₅₀₇ (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY₄₄₇₃ (membrane proximal) bound many fewer proteins and only to the phosphorylated form.     

Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease worldwide. Pathogen adherence to host cells induces reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria. This requires two bacterial virulence factors that mimic a ligand-receptor interaction. EPEC delivers its own receptor, the translocated intimin receptor (Tir), into the target cell plasma membrane, which is phosphorylated on interaction with the bacterial surface protein intimin. Tir phosphorylated on Tyr 474 (ref. 4) binds the cellular adaptor Nck, triggering actin polymerization. Nevertheless, despite its critical role, the mechanism of Tir Tyr 474 phosphorylation remains unknown. Here, by artificially uncoupling Tir delivery and activity, we show that Tir phosphorylation and Nck-dependent pedestal formation require the Src-family kinase (SFK) c-Fyn. SFK inhibitors prevent Tyr 474 phosphorylation, and cells lacking c-fyn are resistant to pedestal formation. c-Fyn exclusively phosphorylates clustered Tir in vitro, and kinase knockdown suppresses Tir phosphorylation and pedestal formation in cultured cells. These results identify the transient interaction with host c-Fyn as a pivotal link between bacterial Tir and the cellular Nck-WASP-Arp2/3 cascade, illuminating a tractable experimental system in which to dissect tyrosine kinase signalling.     

Tyrosine phosphorylation of Sprouty proteins regulates their ability to inhibit growth factor signaling: a dual feedback loop

Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling.     

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.     

Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation,membrane association,and enzyme function in yeast

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1–Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.     

Mutational analysis of stress-responsive peanut dual specificity protein kinase. Identification of tyrosine residues involved in regulation of protein kinase activity

We recently reported that Arachis hypogaea serine/threonine/tyrosine (STY) protein kinase is developmentally regulated and is induced by abiotic stresses (Rudrabhatla, P., and Rajasekharan, R. (2002) Plant Physiol. 130, 380-390). Other than MAPKs, the site of tyrosine phosphorylation has not been documented for any plant kinases. To study the role of tyrosines in the phosphorylation of STY protein kinase, four conserved tyrosine residues were sequentially substituted with phenylalanine and expressed as histidine fusion proteins. Mass spectrometry experiments showed that STY protein kinase autophosphorylated within the predicted kinase ATP-binding motif, activation loop, and an additional site in the C terminus. The protein kinase activity was abolished by substitution of Tyr(297) with Phe in the activation loop between subdomains VII and VIII. In addition, replacing Tyr(148) in the ATP-binding motif and Tyr(317) in the C-terminal domain with Phe not only obliterated the ability of the STY protein kinase protein to be phosphorylated, but also inhibited histone phosphorylation, suggesting that STY protein kinase is phosphorylated at multiple sites. Replacing Tyr(213) in the Thr-Glu-Tyr sequence motif with Phe resulted in a 4-fold increase in autophosphorylation and 2.8-fold increase in substrate phosphorylation activities. Mutants Y148F, Y297F, and Y317F displayed dramatically lower phosphorylation efficiency (k(cat)/K(m)) with ATP and histone, whereas mutant Y213F showed increased phosphorylation. Our results suggest that autophosphorylation of Tyr(148), Tyr(213), Tyr(297), and Tyr(317) is important for the regulation of STY protein kinase activity. Our study reveals the first example of Thr-Glu-Tyr domain-mediated autoinhibition of kinases.     

Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.

In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics.     

Phosphorylation-dependent regulation of Kv2.1 Channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon

Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and up-regulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr(124) is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr(124) is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K(+) currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr(124) is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.     

Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Delta ura8Delta mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Delta ura8Delta mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from (32)P(i)-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.     

Differential dephosphorylation of the FcRgamma immunoreceptor tyrosine-based activation motif tyrosines with dissimilar potential for activating Syk

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.     

Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.The type II secretion (T2S) pathway is widely used by pathogenic gram-negative bacteria for delivery of virulence factors into the extracellular milieu (11, 17, 46). Proteins destined for release through this pathway are first translocated across the cytoplasmic membrane via the Sec (24, 42) or Tat (59) machinery. Following folding and assembly in the periplasm, the proteins are transported across the outer membrane via the T2S machinery, a complex composed of 12 to 16 different gene products, depending on the species. In Vibrio cholerae, the elements of the T2S apparatus are encoded by the extracellular protein secretion (eps) genes, epsC through epsN and pilD (vcpD) (18, 31, 39, 49, 50). Together these proteins coordinate the outer membrane translocation of the major virulence factor, cholera toxin, as well as chitinase, lipase, hemagglutinin/protease, and other proteases (12, 27, 49). Our studies are focused on better understanding how the T2S complex assembles in the cell envelope of V. cholerae to begin to elucidate the mechanism by which extracellular secretion is accomplished.The T2S apparatus is modeled as an envelope-spanning complex with subcomplexes in the inner and outer membranes (see Fig. S1 in the supplemental material). The precise stoichiometry and juxtaposition of the Eps proteins are not known, but accumulating biochemical, genetic, and molecular studies continue to refine our understanding of complex assembly and function (for a review, see reference 25). A trimolecular complex consisting of cytoplasmic protein EpsE and inner membrane proteins EpsL and EpsM has been identified. EpsL and EpsM have been shown to coimmunoprecipitate and participate in mutual stabilization interactions in vivo by protecting each other from proteolysis (34, 41, 43, 48). Homologs of inner membrane protein EpsC have been implicated in interactions with the aforementioned inner membrane subcomplex (20, 29, 57), as well as homologs of outer membrane protein EpsD, which form oligomeric rings through which the secreted substrates, it is hypothesized, exit the cell (1, 10, 36, 38). More specifically, EpsC homologs in Pseudomonas aeruginosa and Klebsiella oxytoca are sensitive to proteolysis or unable to oligomerize in the absence of EpsD homologs (2, 40); however, direct interactions between these two proteins in their full-length forms have not been shown by coimmunoprecipitation or copurification. Although yeast two-hybrid analysis of the periplasmic domains of the Erwinia chrysanthemi EpsC and EpsD homologs also did not reveal interaction (15), recently it was shown that periplasmic subdomains of EpsC and EpsD homologs of Vibrio vulnificus copurified (28). It seems likely that EpsC, having interactions with both inner and outer membrane subcomplexes, plays a crucial role in complex function by connecting the inner membrane components to the outer membrane EpsD pore. Furthermore, it has been speculated that EpsC homologs impart specificity to the various T2S systems by directly interacting with proteins to be secreted (3).We have taken a cell biology approach to characterizing Eps protein interactions, observing the dynamics of green fluorescent protein (GFP)-tagged components of the Eps complex in live cells by fluorescence microscopy. This method permits study of Eps protein assembly in the context of the complete apparatus, situated in both membranes, without the disruptive procedures required for many in vitro molecular and biochemical analyses of protein-protein interactions. Here we present data illustrating the importance of expressing GFP fusions for localization studies with all other interacting components, preserving wild-type stoichiometry and expression levels. In particular, we note that GFP-EpsM does not appear to be focused at the polar membrane as previously described (53), when expressed in balance with its interacting proteins. Chromosomal replacement of epsM and epsC with gfp-tagged versions instead reveals a more distributed pattern, with punctate fluorescent foci along the full length of the cell. We have exploited these chromosomal gfp-eps strains to further dissect the interactions and requirements for localization of EpsC and EpsM by systematically deleting other eps genes in the operon.