scholarly journals Biochemical and Genetic Evidence for Participation of DevR in a Phosphorelay Signal Transduction Pathway Essential for Heterocyst Maturation in Nostoc punctiforme ATCC 29133

1999 ◽  
Vol 181 (14) ◽  
pp. 4430-4434 ◽  
Author(s):  
Kari D. Hagen ◽  
John C. Meeks
Keyword(s):  
Signal Transduction ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Signal Transduction Pathway ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Insertion Mutant ◽  
Nostoc Punctiforme ◽  
Putative Phosphorylation Site

ABSTRACT In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in Nostoc punctiformeATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ. Site-directed mutagenesis was used to generate N. punctiforme mutants with single amino acid substitutions at the putative phosphorylation site of DevR. These mutants exhibited a Fox− phenotype like the original devRinsertion mutant UCD 311, consistent with a phosphotransferase role for DevR.

scholarly journals Genetic Studies of mrp, a Locus Essential for Cellular Aggregation and Sporulation of Myxococcus xanthus

2001 ◽  
Vol 183 (16) ◽  
pp. 4786-4795 ◽  
Author(s):  
Hong Sun ◽  
Wenyuan Shi
Keyword(s):  
Developmental Process ◽  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Regulatory Protein ◽  
Phosphorylation Site ◽  
Receptor Protein ◽  
Insertion Mutant ◽  
Developmental Gene Expression ◽  
Putative Phosphorylation Site ◽  
Cellular Aggregation

ABSTRACT Under starvation conditions, Myxococcus xanthusundergoes a complex developmental process which includes cellular aggregation and sporulation. A transposon insertion mutant (the Tn5-Ω280 mutant) with defects in both aggregation and sporulation was analyzed in this study. The Tn5-Ω280 mutant was found to have a disrupted NtrC-like response regulator designated Myxococcusregulatory protein B (mrpB). Further sequencing analyses revealed a histidine kinase homolog (mrpA) immediately upstream of mrpB and a cyclic AMP receptor protein-like transcriptional regulator (mrpC) downstream ofmrpB. In-frame deletion analyses revealed that both themrpB and mrpC genes were required for cellular aggregation and sporulation but that only mrpAwas required for sporulation only. Site-specific mutagenesis of the putative phosphorylation site of MrpB, D58, showed that a D58A mutation caused defects in both aggregation and sporulation but that a D58E mutation resulted in only a sporulation defect. Further genetic and molecular analyses with reporter genes and reverse transcription-PCR indicated that mrpA and mrpB are cotranscribed but that mrpC is transcribed independently and that all of these genes are developmentally regulated. In addition, MrpB is essential for transcription of mrpC and MrpC regulates its own transcription. These data indicate that Mrp proteins are important components required for M. xanthusdevelopment. The complicated interaction between Mrp proteins may play an important role in regulating developmental gene expression inM. xanthus.

scholarly journals The HrpX/HrpY Two-Component System Activates hrpS Expression, the First Step in the Regulatory Cascade Controlling the Hrp Regulon in Pantoea stewartii subsp. stewartii

2003 ◽  
Vol 16 (3) ◽  
pp. 238-248 ◽  
Author(s):  
Massimo Merighi ◽  
Doris R. Majerczak ◽  
Elizabeth H. Stover ◽  
David L. Coplin
Keyword(s):  
Response Regulator ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
In Planta ◽  
Transcriptional Enhancer ◽  
Pantoea Stewartii ◽  
Regulatory Cascade ◽  
Two Component ◽  
Hrp Genes

A regulatory cascade activating hrp/hrc type III secretion and effector genes was delineated in Pantoea stewartii subsp. stewartii, a bacterial pathogen of corn. Four hrp regulatory genes were characterized: hrpX and hrpY encode the sensor kinase and response regulator, respectively, of a two-component signal transduction system; hrpS encodes an NtrC-like transcriptional enhancer; and hrpL encodes an alternative sigma factor. Epistasis analysis, expression studies using gene fusions, and genetic reconstruction of each step in Escherichia coli were used to delineate the following pathway: HrpY activates hrpS and also positively auto-regulates the hrpXY operon. In turn, HrpS is required for full activation of the σ54-dependent hrpL promoter. Finally, HrpL controls expression of all known hrp and wts genes. In vitro, hrpS and all downstream hrp genes were regulated by pH and salt concentration. Mutants with in-frame deletions in hrpX were still partially virulent on corn but were unable to sense the chemical or metabolic signals that induce hrp genes in vitro. Site-directed mutagenesis of HrpY indicated that aspartate 57 is the probable phosphorylation site and that it is needed for activity. These findings suggest that both HrpX and an alternate mechanism are involved in the activation of HrpY in planta.

scholarly journals A Two-Component Regulatory System Controls Autoregulated Serpin Expression in Bifidobacterium breve UCC2003

2012 ◽  
Vol 78 (19) ◽  
pp. 7032-7041 ◽  
Author(s):  
Pablo Alvarez-Martin ◽  
Mary O'Connell Motherway ◽  
Francesca Turroni ◽  
Elena Foroni ◽  
Marco Ventura ◽  
...  
Keyword(s):  
Response Regulator ◽  
Regulatory System ◽  
Site Directed Mutagenesis ◽  
Insertion Mutant ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Content Type ◽  
Bifidobacterium Breve ◽  
Two Component

ABSTRACTThis work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded byserRK, which is believed to control the expression of theser2003locus inBifidobacterium breveUCC2003. Theser2003locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region ofser2003, and the probable recognition sequence of SerR was determined by a combinatorial approach ofin vitrosite-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of theserRK2CRS in the response ofB. breveto protease-mediated induction was confirmed by generating aB. breve serRinsertion mutant, which was shown to exhibit alteredser2003transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of aB. breve serUmutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

scholarly journals Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

2006 ◽  
Vol 26 (22) ◽  
pp. 8639-8654 ◽  
Author(s):  
Zheng Fu ◽  
Katherine A. Larson ◽  
Raghu K. Chitta ◽  
Sirlester A. Parker ◽  
Benjamin E. Turk ◽  
...  
Keyword(s):  
Germ Cell ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Male Germ Cell ◽  
Intestinal Cell ◽  
Protein Phosphatase 5 ◽  
Extracellular Signal ◽  
Putative Phosphorylation Site ◽  
Yin Yang

ABSTRACT MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position −3 (P−3) being more stringent than for prolines at P−2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT1080S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.

scholarly journals Biochemical Characterization of RssA-RssB, a Two-Component Signal Transduction System Regulating Swarming Behavior in Serratia marcescens

2005 ◽  
Vol 187 (16) ◽  
pp. 5683-5690 ◽  
Author(s):  
Jun-Rong Wei ◽  
Yu-Huan Tsai ◽  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Shang-Chen Hsieh ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Serratia Marcescens ◽  
Biochemical Characterization ◽  
Response Regulator ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Promoter Regions ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37°C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30°C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfFSm ), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

scholarly journals Differential Expression of the CO2 Fixation Operons of Rhodobacter sphaeroides by the Prr/Reg Two-Component System during Chemoautotrophic Growth

2002 ◽  
Vol 184 (23) ◽  
pp. 6654-6664 ◽  
Author(s):  
Janet L. Gibson ◽  
James M. Dubbs ◽  
F. Robert Tabita
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Cytochrome Oxidase ◽  
Rhodobacter Sphaeroides ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Signal Transduction Pathway ◽  
Pentose Phosphate ◽  
Bisphosphate Carboxylase

ABSTRACT In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb I and cbbII promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbbI and cbbII promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbbII promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbbI expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbbI promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.

Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities

1991 ◽  
Vol 11 (12) ◽  
pp. 6016-6025
Author(s):  
X K Zhang ◽  
K N Wills ◽  
M Husmann ◽  
T Hermann ◽  
M Pfahl
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Thyroid Hormone Receptor ◽  
Gene Activation ◽  
Signal Transduction Pathway ◽  
Large Family ◽  
Transduction Pathway ◽  
Regulatory Pathway

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes

1993 ◽  
Vol 13 (9) ◽  
pp. 5659-5669 ◽  
Author(s):  
M Tyers ◽  
B Futcher
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
G1 Phase ◽  
Transduction Pathway ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Factor ◽  
Mitogen Activated Protein

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase.

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