Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein.

The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.     

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.     

Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.     

Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein.

VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.     

Constitutive activation of two-component response regulators: characterization of VirG activation in Agrobacterium tumefaciens

Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.     

VirA of Agrobacterium tumefaciens is an intradimer transphosphorylase and can actively block vir gene expression in the absence of phenolic signals

The VirA-VirG two-component system regulates the 30-gene vir regulon in response to host-released chemical signals. VirA is a homodimeric membrane-spanning histidine protein kinase. Here, we show that mutations in two essential VirA residues, His-474 and Gly-657, can be complemented by the formation of mixed heterodimers, indicating that each subunit of a VirA dimer transphosphorylates the opposite subunit. VirA contains a receiver domain that inhibits kinase activity. We use the forced heterodimer system to show that the two receiver domains of a VirA dimer act independently and that each inhibits the phosphoacceptor subdomain of the opposite subunit. We also demonstrate that merodiploid strains co-expressing constitutive VirA mutants and wild-type VirA show levels of vir gene expression far lower than haploid strains expressing just the constitutive alleles. The fact that wild-type VirA can actively block vir gene expression in the absence of phenolic signals suggests that it might have a phospho-VirG phosphatase activity. The receiver domain of VirA is essential for this activity, whereas residues H474 and G657 of the kinase domain are not required. Merodiploid strains co-expressing a constitutive VirA allele and an allele that is kinase inactive but proficient in the inhibitory activity show strongly inducible vir gene expression, indicating that the inhibitory activity is modulated by environmental signals.     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Cleavage of Shigella surface protein VirG occurs at a specific site, but the secretion is not essential for intracellular spreading.

The large plasmid-encoded outer membrane protein VirG (IcsA) of Shigella flexneri is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that VirG is located at one pole on the surface of the bacterium and secreted into the culture supernatant and that in host cells it is localized along the length of the F-actin tail. The roles of these VirG phenotypes in bacterial spreading still remain to be elucidated. In this study, we examined the surface-exposed portion of the VirG protein by limited trypsin digestion of S. flexneri YSH6000 and determined the sites for VirG processing during secretion into the culture supernatant. Our results indicated that the 85-kDa amino-terminal portion of VirG is located on the external side of the outer membrane, while the 37-kDa carboxy-terminal portion is embedded in it. The VirG cleavage required for release of the 85-kDa protein into the culture supernatant occurred at the Arg-Arg bond at positions 758 to 759. VirG-specific cleavage was observed in Shigella species and enteroinvasive Escherichia coli, which requires an as yet unidentified protease activity governed by the virB gene on the large plasmid. To investigate whether the VirG-specific cleavage occurring in extracellular and intracellular bacteria is essential for VirG function in bacterial spreading, the Arg-Arg cleavage site was modified to an Arg-Asp or Asp-Asp bond. The virG mutants thus constructed were capable of unipolar deposition of VirG on the bacterial surface but were unable to cleave VirG under in vitro or in vivo conditions. However, these mutants were still capable of eliciting aggregation of F-actin at one pole, spreading into adjacent cells, and giving rise to a positive Sereny test. Therefore, the ability to cleave and secrete VirG in Shigella species is not a prerequisite for intracellular spreading.     

Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C.

The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.     

Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.     

Platelet glycoprotein Ib beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.     

Binding of the regulatory protein VirG to the phased signal sequences upstream from virulence genes on the hairy-root-inducing plasmid

The VirG protein is a positive regulator for the virulence genes of which expression is induced by a plant factor, and is essential for Agrobacterium pathogenicity on dicotyledonous plants. The VirG protein of the hairy-root-inducing plasmid A4 was overproduced in Escherichia coli cells, and purified to homogeneity. DNase I footprinting experiments revealed that the purified VirG protein was bound to the upstream region of virulence genes including the phased vir box sequences, which had been presumed to be the VirG recognition signal from the sequence analysis. In dimethyl sulfate footprinting, the VirG protein specifically protected the guanine residues within every vir box sequence. It was concluded that the VirG protein was bound to the phased vir box sequences from the major groove along one side of double-helical DNA.     

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.