Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.     

The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis.

Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.     

Altered phosphorylation of Bacillus subtilis DegU caused by single amino acid changes in DegS.

The Bacillus subtilis sacU locus consists of the degS and degU genes, which play a major role in controlling the production of degradative enzymes including extracellular proteases. DegS has been shown to be autophosphorylated and to transfer the phosphoryl group to DegU. In this study, we partially purified the DegS proteins which carry amino acid changes resulting from various mutations and examined the phosphorylation reaction. The mutations used were degS42, causing a reduction in exoprotease production, and degS100(Hy) and degS200(Hy), causing overproduction of the enzymes. The following results were obtained. The DegS protein derived from degS42 was deficient in both autophosphorylation and subsequent phosphate transfer to DegU. Compared with wild-type DegS, the DegS proteins derived from the overproduction mutations, degS100(Hy) and degS200(Hy), were less active in the autophosphorylation and phosphorylation of DegU. However, the DegU phosphates produced by the mutant DegS proteins were more stable than that produced by the wild-type DegS. These results suggest that phosphorylation is tightly linked to exoprotease production and that the prolonged retention of the phosphoryl moiety on DegU activates the genes for the extracellular proteases. It was also shown that the rate of dephosphorylation of DegU-phosphate was increased as the amount of DegS was increased. All of these results suggest that DegS is involved in the dephosphorylation of DegU-phosphate.     

Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase.

The DegS-DegU protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the DegU protein. Phosphoryl transfer to the wild-type DegU protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the degU24 and degU31 alleles presented a stronger phosphorylation signal compared with that of the wild-type DegU protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the degU24 and degU31 mutants. However, the degU32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified DegU response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the degU32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the degU32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, degU122 and degU146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable DegU proteins, respectively.     

Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation.     

The Pta–AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis

DegU is considered to be an orphan response regulator in Listeria monocytogenes since the gene encoding the cognate histidine kinase DegS is absent from the genome. We have previously shown that DegU is involved in motility, chemotaxis and biofilm formation and contributes to L. monocytogenes virulence. Here, we have investigated the role of DegU phosphorylation in Listeria and shown that DegS of Bacillus subtilis can phosphorylate DegU of L. monocytogenes in vitro. We introduced the B. subtilis degS gene into L. monocytogenes, and showed that this leads to highly increased expression of motility and chemotaxis genes, in a DegU‐dependent fashion. We inactivated the predicted phosphorylation site of DegU by replacing aspartate residue 55 with asparagine and showed that this modified protein (DegU_D55N) is no longer phosphorylated by DegS in vitro. We show that although the unphosphorylated form of DegU retains much of its activity in vivo, expression of motility and chemotaxis genes is lowered in the degU_D55N mutant. We also show that the small‐molecular‐weight metabolite acetyl phosphate is an efficient phosphodonor for DegU in vitro and our evidence suggests this is also true in vivo. Indeed, a L. monocytogenesΔptaΔackA mutant that can no longer synthesize acetyl phosphate was found to be strongly affected in chemotaxis and motility gene expression and biofilm formation. Our findings suggest that phosphorylation by acetyl phosphate could play an important role in modulating DegU activity in vivo, linking its phosphorylation state to the metabolic status of L. monocytogenes.     

Phosphorylation of CpgA protein enhances both its GTPase activity and its affinity for ribosome and is crucial for Bacillus subtilis growth and morphology

In Bacillus subtilis, the ribosome-associated GTPase CpgA is crucial for growth and proper morphology and was shown to be phosphorylated in vitro by the Ser/Thr protein kinase PrkC. To further understand the function of the Escherichia coli RsgA ortholog, CpgA, we first demonstrated that its GTPase activity is stimulated by its association with the 30 S ribosomal subunit. Then the role of CpgA phosphorylation was analyzed. A single phosphorylated residue, threonine 166, was identified by mass spectrometry. Phosphoablative replacement of this residue in CpgA induces a decrease of both its affinity for the 30 S ribosomal subunit and its GTPase activity, whereas a phosphomimetic replacement has opposite effects. Furthermore, cells expressing a nonphosphorylatable CpgA protein present the morphological and growth defects similar to those of a cpgA-deleted strain. Altogether, our results suggest that CpgA phosphorylation on Thr-166 could modulate its ribosome-induced GTPase activity. Given the role of PrkC in B. subtilis spore germination, we propose that CpgA phosphorylation is a key regulatory process that is essential for B. subtilis development.     

Biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius under salt stress conditions

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3-19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 M NaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32 (Hy), which provides for the over-synthesis of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3-19 increased by 6-10 times. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.     

Serine phosphorylation negatively regulates RhoA in vivo

Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.     

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca²⁺-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.     

Phosphorylation of RhoGDI1 by p21-activated kinase 2 mediates basic fibroblast growth factor-stimulated neurite outgrowth in PC12 cells

We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.     

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.     

Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth

Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.     

IKKβ specifically binds to P16 and phosphorylates Ser8 of P16

In spite of its central roles in cell cycle progression, senescence, and aging, knowledge about the posttranslational regulation of P16 (also known as INK4A and MTS1) remains limited. While it has been reported that P16 could be phosphorylated at Ser7, Ser8, Ser140, and Ser152, the corresponding kinases have not been identified yet. Here we report that IKKβ, a primary kinase for IκBα phosphorylation, is involved in P16 phosphorylation. Immunoprecipitation and kinase assays showed that IKKβ specifically binds to P16 and phosphorylates P16 at Ser8 in WI38 cells. Biochemical characterization of phosphomimetic Ser → Glu P16 mutants demonstrated that phosphorylation at Ser8 of P16 brings about a significant loss of its cyclin-dependent kinase (CDK) 4-inhibitory activity while P16 retains structurally and functionally intact upon phosphorylation at Ser7, Ser140, and Ser152. Our results reveal the novel role of IKKβ in P16 phosphorylation and broaden our understanding of the regulation of P16.     

Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU‐P by ClpCP

The response regulator DegU and its cognate kinase DegS constitute a two‐component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU‐lacZ fusions, we showed that phosphorylated DegU (DegU‐P) binds to two direct repeats (DR1 and DR2) of the consensus DegU‐binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATP‐dependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild‐type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU‐P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU‐P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N‐terminal region of DegU is important for proteolysis.     

Chaperone Hsp27 modulates AUF1 proteolysis and AU-rich element-mediated mRNA degradation

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues--Ser(15), Ser(78), and Ser(82)-by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2-Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization.