Journal articles: 'Proteine phos'

1

Vogel, K., W. Hörz, and A. Hinnen. "The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions." Molecular and Cellular Biology 9, no. 5 (May 1989): 2050–57. http://dx.doi.org/10.1128/mcb.9.5.2050.

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The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.

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Vogel, K., W. Hörz, and A. Hinnen. "The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions." Molecular and Cellular Biology 9, no. 5 (May 1989): 2050–57. http://dx.doi.org/10.1128/mcb.9.5.2050-2057.1989.

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The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.

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Barbaric, Slobodan, Martin Münsterkötter, Colin Goding, and Wolfram Hörz. "Cooperative Pho2-Pho4 Interactions at thePHO5 Promoter Are Critical for Binding of Pho4 to UASp1 and for Efficient Transactivation by Pho4 at UASp2." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2629–39. http://dx.doi.org/10.1128/mcb.18.5.2629.

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ABSTRACT The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes toPHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

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Pondugula, Santhi, Daniel W. Neef, Warren P. Voth, Russell P. Darst, Archana Dhasarathy, M. Megan Reynolds, Shinya Takahata, David J. Stillman, and Michael P. Kladde. "Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins." Molecular and Cellular Biology 29, no. 18 (July 13, 2009): 4891–905. http://dx.doi.org/10.1128/mcb.00222-09.

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ABSTRACT Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G2/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G1. These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G1 induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

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Antelmann, Haike, Christian Scharf, and Michael Hecker. "Phosphate Starvation-Inducible Proteins ofBacillus subtilis: Proteomics and Transcriptional Analysis." Journal of Bacteriology 182, no. 16 (August 15, 2000): 4478–90. http://dx.doi.org/10.1128/jb.182.16.4478-4490.2000.

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ABSTRACT The phosphate starvation response in Bacillus subtiliswas analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of ςB, the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and aphoR mutant. By matrix-assisted laser desorption ionization–time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhFconfirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a ςA promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed withphoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in bothphoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by ςB-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a ςB-dependent manner and in the sigB mutant probably via ςH. YxiE was induced by phosphate starvation independently of ςB and PhoPR.

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Teng, Fang, Ling Wang, Kavindra V. Singh, Barbara E. Murray, and George M. Weinstock. "Involvement of PhoP-PhoS Homologs in Enterococcus faecalis Virulence." Infection and Immunity 70, no. 4 (April 2002): 1991–96. http://dx.doi.org/10.1128/iai.70.4.1991-1996.2002.

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ABSTRACT Eleven PhoP-PhoS homolog pairs were identified by searching the Enterococcus faecalis V583 genome sequence database at The Institute for Genomic Research with the Bacillus subtilis PhoP-PhoS sequences. Each pair appears to be a potential two-component system composed of a response regulator and a sensor kinase. Seven of the homologs were disrupted in E. faecalis strain OG1RF. TX10293, a mutant disrupted in one of these genes (etaR, the first gene of the gene pair designated etaRS), showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. The predicted EtaR protein sequence showed greatest similarity to LisR of Listeria monocytogenes (77%) and CsrR of Streptococcus pyogenes (70%); EtaS is 53% similar to LisK and 54% similar to CsrS. When grown in vitro, the TX10293 mutant was more sensitive to low pH (pH 3.4) and more resistant to high temperature (55°C) than wild-type OG1RF. In conclusion, many potential two-component systems are identified for E. faecalis, one of which, EtaRS, was shown to be involved in stress response and virulence.

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Barbaric, S., M. Munsterkotter, J. Svaren, and W. Horz. "The Homeodomain Protein Pho2 and the Basic-Helix-Loop-Helix Protein Pho4 Bind DNA Cooperatively at the Yeast PHO5 Promoter." Nucleic Acids Research 24, no. 22 (November 1, 1996): 4479–86. http://dx.doi.org/10.1093/nar/24.22.4479.

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8

Ogawa, N., and Y. Oshima. "Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 5 (May 1990): 2224–36. http://dx.doi.org/10.1128/mcb.10.5.2224.

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The PHO4 gene encodes a positive regulatory factor involved in regulating transcription of various genes in the phosphatase regulon of Saccharomyces cerevisiae. Besides its own coding region, the 1.8-kilobase PHO4 transcript contains a coding region for a mitochondrial protein which does not appear to be translated. Four functional domains were found in the PHO4 protein, which consists of 312 amino acid (aa) residues as deduced from the open reading frame of PHO4. A gel retardation assay with beta-galactosidase::PHO4 fused protein revealed that the 85-aa C terminus is the domain responsible for binding to the promoter DNA of PHO5, a gene under the control of PHO4. This region has similarities with the amphipathic helix-loop-helix motif of c-myc protein. Determination of the nucleotide sequences of four PHO4c mutant alleles and insertion and deletion analyses of PHO4 DNA indicated that a region from aa 163 to 202 is involved in interaction with a negative regulatory factor PHO80. Complementation of a pho4 null allele with the modified PHO4 DNAs suggested that the N-terminal region (1 to 109 aa), which is rich in acidic aa, is the transcriptional activation domain. The deleterious effects of various PHO4 mutations on the constitutive transcription of PHO5 in PHO4c mutant cells suggested that the region from aa 203 to 227 is involved in oligomerization of the PHO4 protein.

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9

Ogawa, N., and Y. Oshima. "Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 5 (May 1990): 2224–36. http://dx.doi.org/10.1128/mcb.10.5.2224-2236.1990.

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The PHO4 gene encodes a positive regulatory factor involved in regulating transcription of various genes in the phosphatase regulon of Saccharomyces cerevisiae. Besides its own coding region, the 1.8-kilobase PHO4 transcript contains a coding region for a mitochondrial protein which does not appear to be translated. Four functional domains were found in the PHO4 protein, which consists of 312 amino acid (aa) residues as deduced from the open reading frame of PHO4. A gel retardation assay with beta-galactosidase::PHO4 fused protein revealed that the 85-aa C terminus is the domain responsible for binding to the promoter DNA of PHO5, a gene under the control of PHO4. This region has similarities with the amphipathic helix-loop-helix motif of c-myc protein. Determination of the nucleotide sequences of four PHO4c mutant alleles and insertion and deletion analyses of PHO4 DNA indicated that a region from aa 163 to 202 is involved in interaction with a negative regulatory factor PHO80. Complementation of a pho4 null allele with the modified PHO4 DNAs suggested that the N-terminal region (1 to 109 aa), which is rich in acidic aa, is the transcriptional activation domain. The deleterious effects of various PHO4 mutations on the constitutive transcription of PHO5 in PHO4c mutant cells suggested that the region from aa 203 to 227 is involved in oligomerization of the PHO4 protein.

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10

Allen, Mindy P., Kimberly B. Zumbrennen, and William R. McCleary. "Genetic Evidence that the α5 Helix of the Receiver Domain of PhoB Is Involved in Interdomain Interactions." Journal of Bacteriology 183, no. 7 (April 1, 2001): 2204–11. http://dx.doi.org/10.1128/jb.183.7.2204-2211.2001.

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ABSTRACT Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein. Generally, response regulators are made up of a receiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain. The mechanisms by which receiver domains control the activities of their respective output domains are unknown. To address this question for the PhoB protein from Escherichia coli, we have employed two separate genetic approaches, deletion analysis and domain swapping. In-frame deletions were generated within the phoB gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the phoA gene. However, another deletion mutant that contained the C-terminal α-helix of the receiver domain (α5) in addition to the entire output domain was unable to activate transcription of phoA. This result suggests that the α5 helix of the receiver domain interacts with and inhibits the output domain. We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N-terminal ∼85% of CheY's receiver domain to the β5-α5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the α5 helix of its unphosphorylated receiver domain and its output domain.

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Hayashi, N., and Y. Oshima. "Specific cis-acting sequence for PHO8 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 2 (February 1991): 785–94. http://dx.doi.org/10.1128/mcb.11.2.785.

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The PHO8 gene of Saccharomyces cerevisiae encodes repressible alkaline phosphatase (rALPase; EC 3.1.3.1). The rALPase activity of the cells is two to three times higher in medium containing a low concentration of Pi than in high-Pi medium due to transcription of PHO8. The Pi signals are conveyed to PHO8 by binding of PHO4 protein, a positive regulatory factor, to a promoter region of PHO8 (PHO8p) under the influence of the PHO regulatory circuit. Deletion analysis of PHO8p DNA revealed two separate regulatory regions required for derepression of rALPase located at nucleotide positions -704 to -661 (distal region) and -548 to -502 (proximal region) and an inhibitory region located at -421 to -289 relative to the translation initiation codon. Gel retardation experiments showed that a beta-galactosidase-PHO4 fusion protein binds to a 132-bp PHO8p fragment bearing the proximal region but not to a 226-bp PHO8 DNA bearing the distal region. The fusion protein also binds to a synthetic oligonucleotide having the same 12-bp nucleotide sequence as the PHO8p DNA from positions -536 to -525. The 132-bp PHO8p fragment, connected at position -281 of the 5' upstream region of a HIS5'-'lacZ fused gene, could sense Pi signals in vivo, but a 20-bp synthetic oligonucleotide having the same sequence from -544 to -525 of the PHO8p DNA could not. Linker insertions in the PHO8p DNA indicated that the 5-bp sequence 5'-CACGT-3' from positions -535 to -531 is essential for binding the beta-galactosidase-PHO4 fusion protein and for derepression of rALPase.

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Hayashi, N., and Y. Oshima. "Specific cis-acting sequence for PHO8 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 2 (February 1991): 785–94. http://dx.doi.org/10.1128/mcb.11.2.785-794.1991.

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The PHO8 gene of Saccharomyces cerevisiae encodes repressible alkaline phosphatase (rALPase; EC 3.1.3.1). The rALPase activity of the cells is two to three times higher in medium containing a low concentration of Pi than in high-Pi medium due to transcription of PHO8. The Pi signals are conveyed to PHO8 by binding of PHO4 protein, a positive regulatory factor, to a promoter region of PHO8 (PHO8p) under the influence of the PHO regulatory circuit. Deletion analysis of PHO8p DNA revealed two separate regulatory regions required for derepression of rALPase located at nucleotide positions -704 to -661 (distal region) and -548 to -502 (proximal region) and an inhibitory region located at -421 to -289 relative to the translation initiation codon. Gel retardation experiments showed that a beta-galactosidase-PHO4 fusion protein binds to a 132-bp PHO8p fragment bearing the proximal region but not to a 226-bp PHO8 DNA bearing the distal region. The fusion protein also binds to a synthetic oligonucleotide having the same 12-bp nucleotide sequence as the PHO8p DNA from positions -536 to -525. The 132-bp PHO8p fragment, connected at position -281 of the 5' upstream region of a HIS5'-'lacZ fused gene, could sense Pi signals in vivo, but a 20-bp synthetic oligonucleotide having the same sequence from -544 to -525 of the PHO8p DNA could not. Linker insertions in the PHO8p DNA indicated that the 5-bp sequence 5'-CACGT-3' from positions -535 to -531 is essential for binding the beta-galactosidase-PHO4 fusion protein and for derepression of rALPase.

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Nishioka, Keiji, Yusuke Kato, Shin-ichiro Ozawa, Yuichiro Takahashi, and Wataru Sakamoto. "Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane." Photosynthesis Research 147, no. 1 (December 2, 2020): 107–24. http://dx.doi.org/10.1007/s11120-020-00803-1.

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AbstractProtein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

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Tibes, Raoul, YiHua Qiu, Kevin R. Coombes, David Gold, Gordon B. Mills, and Steven M. Kornblau. "Classification of Acute Myelogenous Leukemia (AML) Based on Patterns of Signal Transduction Pathway (STP) and Apoptosis Regulating Protein Activation Determined by Reverse Phase Proteins Arrays (RPPA)." Blood 106, no. 11 (November 16, 2005): 484. http://dx.doi.org/10.1182/blood.v106.11.484.484.

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Abstract We have previously shown that the protein expression profile and especially the activation state of apoptosis and STP proteins are highly prognostic in AML. The development of RPPA permits a more comprehensive analysis of protein expression and phosphorylation (phos) patterns in the STP and apoptotic cascades. We have generated a screening array using protein derived from the leukemia enriched fraction of 94 primary AML samples (with 21 concurrent blood and marrow specimens), comprised equally of patients that were primary refractory (PrimRef), or that achieved complete remission that was continuous (CCR) or which relapsed after 6 to 24 months (REL). These slides have been probed with 22 total and 15 phos-specific antibodies (ABs) against apoptosis and STP proteins. Spot intensities were quantified using MicroVigene and the data for each protein standardized data by subtracting the mean expression levels across samples and dividing by the standard deviation. Unsupervised hierarchical clustering analysis on the proteins was performed. We found one small cluster of 5 proteins: Cyclin D1, β-catenin, phos-NMP, p53, and AMPK. The remaining proteins formed another cluster, with a subset of 8 proteins somewhat further separated. In several cases (mTOR, JNK, and PTEN) the total protein amounts clustered as nearest neighbors to the phos version of the same protein. Using perturbation bootstrap resampling to assess the significance of clusters we found 4 reproducible clusters and the structure suggests additional clusters in this data set. A Fisher exact test showed that these were significantly associated with response (p = 0.03) and with the prognostic category defined by cytogenetics (p = 0.04) but was not associated with the source (blood vs. marrow) of the sample (p = 0.67). Ten proteins were differentially expressed between cytogenetically defined prognostic groups: total and phospho AKT, PTEN and JNK, p-mTOR, p-STAT3, pp38 and PS6.p24.44 (all with P<0.02). No protein was prognostic for response across the entire population but several were prognostic for response and survival within specific clusters. Clustering of ratios of phos-/total AB revealed 2 patterns: one with higher phos- of kinases (“kinase high”); another with low kinase expression (“kinase low”) but higher phos- of apoptosis regulating proteins. In the “kinase high” cluster single or concurrent increased activation of pAkt308, pAkt473, pPKCa, p-mTor (2448), pErk1,2 (42/44), p-p38 over their corresponding total protein was associated with worse outcome (~15% CCR rate). Those with increased Bcl2/Bax and pJNK/JNK levels fared worse with only 10% CCR. A favorable outcome was associated with concurrent high pAkt308/Akt and pGSK3/GSK3 levels (50% CCR) or being in the “kinase low” group while having high pStat3(705)/Stat3 levels (58% CCRs). The identified clusters and their correlation with cytogenetic group and outcome mainly driven by the activated, phospho protein forms, strongly supports the idea that the activation state of STP and apoptotic regulators determines biological behavior and clinical outcome. Distinct pathway activation patterns can classify AML, provide prognostic guidance and may serve to triage patients to emerging targeted therapies aimed at these pathways.

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Yu, Guowu, Yanan Lv, Leiyang Shen, Yongbin Wang, Yun Qing, Nan Wu, Yangping Li, et al. "The Proteomic Analysis of Maize Endosperm Protein Enriched by Phos-tagtm Reveals the Phosphorylation of Brittle-2 Subunit of ADP-Glc Pyrophosphorylase in Starch Biosynthesis Process." International Journal of Molecular Sciences 20, no. 4 (February 24, 2019): 986. http://dx.doi.org/10.3390/ijms20040986.

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AGPase catalyzes a key rate-limiting step that converts ATP and Glc-1-p into ADP-glucose and diphosphate in maize starch biosynthesis. Previous studies suggest that AGPase is modulated by redox, thermal and allosteric regulation. However, the phosphorylation of AGPase is unclear in the kernel starch biosynthesis process. Phos-tagTM technology is a novel method using phos-tagTM agarose beads for separation, purification, and detection of phosphorylated proteins. Here we identified phos-tagTM agarose binding proteins from maize endosperm. Results showed a total of 1733 proteins identified from 10,678 distinct peptides. Interestingly, a total of 21 unique peptides for AGPase sub-unit Brittle-2 (Bt2) were identified. Bt2 was demonstrated by immunoblot when enriched maize endosperm protein with phos-tagTM agarose was in different pollination stages. In contrast, Bt2 would lose binding to phos-tagTM when samples were treated with alkaline phosphatase (ALP). Furthermore, Bt2 could be detected by Pro-Q diamond staining specifically for phosphorylated protein. We further identified the phosphorylation sites of Bt2 at Ser10, Thr451, and Thr462 by iTRAQ. In addition, dephosphorylation of Bt2 decreased the activity of AGPase in the native gel assay through ALP treatment. Taking together, these results strongly suggest that the phosphorylation of AGPase may be a new model to regulate AGPase activity in the starch biosynthesis process.

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Minh Thu, Doan, Nguyen Thi Minh Viet, and Pham Thi Kim Lien. "Detection of protein stoichiometric phosphorylation using Phos-tag SDS-PAGE." Vietnam Journal of Biotechnology 17, no. 4 (November 2, 2020): 645–49. http://dx.doi.org/10.15625/1811-4989/17/4/13785.

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Protein phosphorylation plays an important role in many cellular signalings which are relating to many diseases. Therefore, a variety of biochemical techniques has been developed to study protein phosphorylation in cells. Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, these antibodies cannot be used to detect unidentified phosphorylation sites. Recently, the Phos-tag technology has been developed to overcome the disadvantages and limitations of these methods. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, and can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. The grouping of the Phos-tag will alter the mobility of protein on the gel depending on the amount of serine, threonine or tyrosine which are phosphorylated. Here, we describe the method to detect the phosphorylation of Pop2 protein, one of the exonucleases in the Ccr4-Not complex regulating the shortening of poly(A) tail of mRNAs using phosphate affinity Phos-tag SDS-PAGE. We observed clear electrophoretic 04 shift bands of Pop2-3XFlag under unstressed conditions. This is the first study which observes Pop2 phosphorylation in normal culture conditions. This study showed the convenience and advantages of Phos-tag SDS-PAGE for research on molecular mechanisms regulating the function of protein.

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Bardin, Sylvie D., and Turlough M. Finan. "Regulation of Phosphate Assimilation in Rhizobium (Sinorhizobium) meliloti." Genetics 148, no. 4 (April 1, 1998): 1689–700. http://dx.doi.org/10.1093/genetics/148.4.1689.

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Abstract We report the isolation of phoB and phoU mutants of the bacterium Rhizobium (Sinorhizobium) meliloti. These mutants form N2-fixing nodules on the roots of alfalfa plants. R. meliloti mutants defective in the phoCDET (ndvF) encoded phosphate transport system grow slowly in media containing 2 mm Pi, and form nodules which fail to fix nitrogen (Fix−). We show that the transfer of phoB or phoU insertion mutations into phoC mutant strains restores the ability of these mutants to: (i) form normal N2-fixing root-nodules, and (ii) grow like the wild type in media containing 2 mm Pi. We also show that expression of the alternate orfA pit encoded Pi transport system is negatively regulated by the phoB gene product, whereas phoB is required for phoCDET expression. We suggest that in R. meliloti cells growing under Pi limiting conditions, PhoB protein activates phoCDET transcription and represses orfA pit transcription. Our results suggest that there are major differences between the Escherichia coli and R. meliloti phosphate regulatory systems.

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Fan, Lihong, Jia Li, Zefeng Yu, Xiaoqian Dang, and Kunzheng Wang. "The Hypoxia-Inducible Factor Pathway, Prolyl Hydroxylase Domain Protein Inhibitors, and Their Roles in Bone Repair and Regeneration." BioMed Research International 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/239356.

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Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators that play crucial roles in angiogenesis, erythropoiesis, energy metabolism, and cell fate decisions. The group of enzymes that can catalyse the hydroxylation reaction of HIF-1 is prolyl hydroxylase domain proteins (PHDs). PHD inhibitors (PHIs) activate the HIF pathway by preventing degradation of HIF-αvia inhibiting PHDs. Osteogenesis and angiogenesis are tightly coupled during bone repair and regeneration. Numerous studies suggest that HIFs and their target gene, vascular endothelial growth factor (VEGF), are critical regulators of angiogenic-osteogenic coupling. In this brief perspective, we review current studies about the HIF pathway and its role in bone repair and regeneration, as well as the cellular and molecular mechanisms involved. Additionally, we briefly discuss the therapeutic manipulation of HIFs and VEGF in bone repair and bone tumours. This review will expand our knowledge of biology of HIFs, PHDs, PHD inhibitors, and bone regeneration, and it may also aid the design of novel therapies for accelerating bone repair and regeneration or inhibiting bone tumours.

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O'Connell, K. F., and R. E. Baker. "Possible cross-regulation of phosphate and sulfate metabolism in Saccharomyces cerevisiae." Genetics 132, no. 1 (September 1, 1992): 63–73. http://dx.doi.org/10.1093/genetics/132.1.63.

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Abstract CP1 (encoded by the gene CEP1) is a sequence-specific DNA binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 exhibit defects in growth, chromosome segregation and methionine biosynthesis. A YEp24-based yeast genomic library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80 and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was PHO2-dependent. Spontaneously arising extragenic suppressors of cep1 methionine auxotrophy were also isolated; approximately one-third of them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low inorganic phosphate growth deficiency of a pho84 mutant. The results may suggest that phosphate and sulfate metabolism are cross-regulated.

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Amemura, M., K. Makino, H. Shinagawa, and A. Nakata. "Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2." Journal of Bacteriology 172, no. 11 (1990): 6300–6307. http://dx.doi.org/10.1128/jb.172.11.6300-6307.1990.

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Kageyama, Hakuto, Keshawanand Tripathi, Ashwani K. Rai, Suriyan Cha-um, Rungaroon Waditee-Sirisattha, and Teruhiro Takabe. "An Alkaline Phosphatase/Phosphodiesterase, PhoD, Induced by Salt Stress and Secreted Out of the Cells of Aphanothece halophytica, a Halotolerant Cyanobacterium." Applied and Environmental Microbiology 77, no. 15 (June 10, 2011): 5178–83. http://dx.doi.org/10.1128/aem.00667-11.

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ABSTRACTAlkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned thephoDgene from a halotolerant cyanobacterium,Aphanothece halophytica(phoDAp). The deduced protein, PhoDAp, contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoDApenzyme was activated by Ca2+and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoDApin a transformed cyanobacterium. Expression of thephoDApgene inA. halophyticacells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest thatA. halophyticacells possess a PhoD that participates in the assimilation of P under salinity stress.

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van Velkinburgh, Jennifer C., and John S. Gunn. "PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp." Infection and Immunity 67, no. 4 (April 1, 1999): 1614–22. http://dx.doi.org/10.1128/iai.67.4.1614-1622.1999.

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ABSTRACT As enteric pathogens, Salmonella spp. are resistant to the actions of bile. Salmonella typhimurium andSalmonella typhi strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-typeS. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% bile dramatically increased bile resistance. Mutant strains of S. typhimurium andS. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC andprgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder.

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Choi, Eunna, Eduardo A. Groisman, and Dongwoo Shin. "Activated by Different Signals, the PhoP/PhoQ Two-Component System Differentially Regulates Metal Uptake." Journal of Bacteriology 191, no. 23 (October 2, 2009): 7174–81. http://dx.doi.org/10.1128/jb.00958-09.

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ABSTRACT The PhoP/PhoQ two-component system controls several physiological and virulence functions in Salmonella enterica. This system is activated by low Mg2+, acidic pH, and antimicrobial peptides, but the biological consequences resulting from sensing multiple signals are presently unclear. Here, we report that the PhoP/PhoQ system regulates different Salmonella genes depending on whether the inducing signal is acidic pH or low Mg2+. When Salmonella experiences acidic pH, the PhoP/PhoQ system promotes Fe2+ uptake in a process that requires the response regulator RstA, activating transcription of the Fe2+ transporter gene feoB. In contrast, the PhoP-induced RstA protein did not promote feoB expression at neutral pH with low Mg2+. The PhoP/PhoQ system promotes the expression of the Mg2+ transporter mgtA gene only when activated in bacteria starved for Mg2+. This is because mgtA transcription promoted at high Mg2+ concentrations by the acidic-pH-activated PhoP protein failed to reach the mgtA coding region due to the mgtA leader region functioning as a Mg2+ sensor. Our results show that a single two-component regulatory system can regulate distinct sets of genes in response to different input signals.

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Zhang, Y., L. Wang, Y. Han, Y. Yan, Y. Tan, L. Zhou, Y. Cui, et al. "Autoregulation of PhoP/PhoQ and Positive Regulation of the Cyclic AMP Receptor Protein-Cyclic AMP Complex by PhoP in Yersinia pestis." Journal of Bacteriology 195, no. 5 (December 21, 2012): 1022–30. http://dx.doi.org/10.1128/jb.01530-12.

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Nagai, Shigeki, Ralph E. Davis, Pierre Jean Mattei, Kyle Patrick Eagen, and Roger D. Kornberg. "Chromatin potentiates transcription." Proceedings of the National Academy of Sciences 114, no. 7 (January 30, 2017): 1536–41. http://dx.doi.org/10.1073/pnas.1620312114.

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Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.

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Salazar, Michael E., Anna I. Podgornaia, and Michael T. Laub. "The small membrane protein MgrB regulates PhoQ bifunctionality to control PhoP target gene expression dynamics." Molecular Microbiology 102, no. 3 (August 25, 2016): 430–45. http://dx.doi.org/10.1111/mmi.13471.

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Davido, David J., William F. von Zagorski, William S. Lane, and Priscilla A. Schaffer. "Phosphorylation Site Mutations Affect Herpes Simplex Virus Type 1 ICP0 Function." Journal of Virology 79, no. 2 (January 15, 2005): 1232–43. http://dx.doi.org/10.1128/jvi.79.2.1232-1243.2005.

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ABSTRACT The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.

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Kim, Jinoh, and Debra A. Kendall. "Identification of a Sequence Motif That Confers SecB Dependence on a SecB-Independent Secretory Protein In Vivo." Journal of Bacteriology 180, no. 6 (March 15, 1998): 1396–401. http://dx.doi.org/10.1128/jb.180.6.1396-1401.1998.

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ABSTRACT SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, inEscherichia coli. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.

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ISHII, Eiji, Yoko EGUCHI, and Ryutaro UTSUMI. "Mechanism of Activation of PhoQ/PhoP Two-Component Signal Transduction by SafA, an Auxiliary Protein of PhoQ Histidine Kinase in Escherichia coli." Bioscience, Biotechnology, and Biochemistry 77, no. 4 (April 23, 2013): 814–19. http://dx.doi.org/10.1271/bbb.120970.

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Makino, Kozo, Hideo Shinagawa, Mitsuko Amemura, Takeshi Kawamoto, Masami Yamada, and Atsuo Nakata. "Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins." Journal of Molecular Biology 210, no. 3 (December 1989): 551–59. http://dx.doi.org/10.1016/0022-2836(89)90131-9.

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Barbaric, Slobodan, Hans Reinke, and Wolfram Hörz. "Multiple Mechanistically Distinct Functions of SAGA at the PHO5 Promoter." Molecular and Cellular Biology 23, no. 10 (May 15, 2003): 3468–76. http://dx.doi.org/10.1128/mcb.23.10.3468-3476.2003.

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ABSTRACT Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity. Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter. Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions. Spt3, another subunit of SAGA, also plays a role in PHO5 activation but has a function that is completely different from that of Gcn5. An SPT3 deletion severely compromises the PHO5 promoter and reduces the extent of transcriptional activation by diminishing the binding of the TATA binding protein to the promoter without, however, affecting the rate or the extent of chromatin remodeling. A gcn5 spt3 double mutant shows a synthetic phenotype almost as severe as that observed for an spt7 or spt20 mutant. The latter two mutations are known to prevent the assembly of the complex and consequently lead to the loss of all SAGA functions. The absence of the Ada2 subunit causes a strong delay in chromatin remodeling and promoter activation that closely resembles the delay observed in the absence of Gcn5. A deletion of only the Ada2 SANT domain has exactly the same effect, strongly suggesting that Ada2 controls Gcn5 activity by virtue of its SANT domain. Finally, the Gcn5 bromodomain also contributes to but is not essential for Gcn5 function at the PHO5 promoter. Taken together, the results provide a detailed and differentiated description of the role of SAGA as a coactivator at the PHO5 promoter.

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Pegues, David A., Michael J. Hantman, Irmgard Behlau, and Samuel I. Miller. "PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion." Molecular Microbiology 17, no. 1 (July 1995): 169–81. http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_17010169.x.

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Long, W., L. Saffer, L. Wei, and E. J. Barrett. "Amino acids regulate skeletal muscle PHAS-I and p70 S6-kinase phosphorylation independently of insulin." American Journal of Physiology-Endocrinology and Metabolism 279, no. 2 (August 1, 2000): E301—E306. http://dx.doi.org/10.1152/ajpendo.2000.279.2.e301.

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Refeeding reverses the muscle protein loss seen with fasting. The physiological regulators and cellular control sites responsible for this reversal are incompletely defined. Phosphorylation of phosphorylated heat-acid stabled protein (PHAS-I) frees eukaryotic initiation factor 4E (eIF4E) and stimulates protein synthesis by accelerating translation initiation. Phosphorylation of p70 S6-kinase (p70S6k) is thought to be involved in the regulation of the synthesis of some ribosomsal proteins and other selected proteins with polypyrimidine clusters near the transcription start site. We examined whether phosphorylation of PHAS-I and p70S6k was increased by feeding and determined the separate effects of insulin and amino acids on PHAS-I and p70S6k phosphorylation in rat skeletal muscle in vivo. Muscle was obtained from rats fed ad libitum or fasted overnight ( n = 5 each). Other fasted rats were infused with insulin (3 μU · min−1 · kg−1, euglycemic clamp), amino acids, or the two combined. Gastrocnemius was freeze-clamped, and PHAS-I and p70S6k phosphorylation was measured by quantifying the several phosphorylated forms of these proteins seen on Western blots. We observed that feeding increased phosphorylation of both PHAS-I and p70S6k ( P < 0.05). Infusion of amino acids alone reproduced the effect of feeding. Physiological hyperinsulinemia increased p70S6K ( P< 0.05) but not PHAS-I phosphorylation ( P = 0.98). Addition of insulin to amino acid infusion was no more effective than amino acids alone in promoting PHAS-I and p70S6kphosphorylation. We conclude that amino acid infusion alone enhances the activation of the protein synthetic pathways in vivo in rat skeletal muscle. This effect is not dependent on increases in plasma insulin and simulates the activation of protein synthesis that accompanies normal feeding.

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Zhou, Lu, Gérald Grégori, Jennifer Masella Blackman, J. Paul Robinson, and Barry L. Wanner. "Stochastic activation of the response regulator PhoB by noncognate histidine kinases." Journal of Integrative Bioinformatics 2, no. 1 (December 1, 2005): 10–22. http://dx.doi.org/10.1515/jib-2005-11.

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Abstract Two-component systems (TCS) are the most prevalent gene regulatory mechanism in bacteria. A typical TCS is comprised of a histidine kinase (HK) and a partner response regulator (RR). Specific environment signals lead to autophosphorylation of different HKs, which in turn act as phosphoryl donors for autophosphorylation of their partner RRs. Nonpartner HKs and RRs also interact, giving rise to cross regulation among TCSs in response to diverse signals.PhoR (HK) and PhoB (RR) constitute the TCS for detection of environmental (extracellular) inorganic phosphate (Pi). The PhoR/PhoB TCS controls the expression of a large number of genes for acquisition of alternative phosphorus sources, including phoA, which encodes the non-specific phosphohydrolase bacterial alkaline phosphatase (Bap). Cross activation of PhoB by the nonpartner HK CreC is now a classic example of cross regulation among TCSs. A systematic search for other cross talking HKs revealed five additional HKs that activate (phosphorylate) PhoB (J. M. B. and B. L. W., unpublished data).Examination of cross activation of PhoB by these non-partner HKs by flow cytometry at the single-cell level revealed a bimodal, “all-or-none,” distribution pattern for expression of a phoAp-gfp (green fluorescent protein) reporter fusion. Although the basis of the observed stochastic behavior is unclear, it seems to reflect an inherent property of TCSs. We propose that cells exploit the stochastic character of TCSs to achieve nongenetic (epigenetic) diversity within genetically homogeneous cell populations in order to facilitate adaptation to environmental changes.

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Ismail, Hamid D., Ahoi Jones, Jung H. Kim, Robert H. Newman, and Dukka B. KC. "RF-Phos: A Novel General Phosphorylation Site Prediction Tool Based on Random Forest." BioMed Research International 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/3281590.

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Protein phosphorylation is one of the most widespread regulatory mechanisms in eukaryotes. Over the past decade, phosphorylation site prediction has emerged as an important problem in the field of bioinformatics. Here, we report a new method, termed Random Forest-based Phosphosite predictor 2.0 (RF-Phos 2.0), to predict phosphorylation sites given only the primary amino acid sequence of a protein as input. RF-Phos 2.0, which uses random forest with sequence and structural features, is able to identify putative sites of phosphorylation across many protein families. In side-by-side comparisons based on 10-fold cross validation and an independent dataset, RF-Phos 2.0 compares favorably to other popular mammalian phosphosite prediction methods, such as PhosphoSVM, GPS2.1, and Musite.

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Lemire, J. M., T. Willcocks, H. O. Halvorson, and K. A. Bostian. "Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 5, no. 8 (August 1985): 2131–41. http://dx.doi.org/10.1128/mcb.5.8.2131.

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We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.

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Lemire, J. M., T. Willcocks, H. O. Halvorson, and K. A. Bostian. "Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 5, no. 8 (August 1985): 2131–41. http://dx.doi.org/10.1128/mcb.5.8.2131-2141.1985.

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We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.

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Runyen-Janecky, L. J., A. M. Boyle, A. Kizzee, L. Liefer, and S. M. Payne. "Role of the Pst System in Plaque Formation by the Intracellular Pathogen Shigella flexneri." Infection and Immunity 73, no. 3 (March 2005): 1404–10. http://dx.doi.org/10.1128/iai.73.3.1404-1410.2005.

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ABSTRACT In response to the host cell environment, the intracellular pathogen Shigella flexneri induces the expression of numerous genes, including those in the pst operon which is predicted to encode a high-affinity phosphate acquisition system that is expressed under reduced phosphate conditions. An S. flexneri pst mutant forms smaller plaques in Henle cell monolayers than does the parental strain. This mutant exhibited normal production and localization of the S. flexneri IcsA protein. The pst mutant had the same growth rate as the parental strain in both phosphate-reduced and phosphate-replete media in vitro and during the first 3 h of growth in Henle cells in vivo. During growth in phosphate-replete media, the PhoB regulon was constitutively expressed in the pst mutant but not the parental strain. This suggested that the inability of the S. flexneri pst mutant to form wild-type plaques in Henle cell monolayers may be due to aberrant expression of the PhoB regulon. A mutation in phoB was constructed in the S. flexneri pst mutant, and the phoB mutation suppressed the small plaque phenotype of the pst mutant. Additionally, a specific mutation (R220Q) was constructed in the pstA gene of the pst operon that was predicted to eliminate Pst-mediated phosphate transport but allow normal PhoB-regulated gene expression, based on the phenotype of an Escherichia coli strain harboring the same mutation. Addition of this pstA R220Q mutation to a S. flexneri pst mutant, as part of the pst operon, restored normal plaque formation and regulation of phoA expression.

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Gusa, Asiya A., Jinxin Gao, Virginia Stringer, Gordon Churchward, and June R. Scott. "Phosphorylation of the Group A Streptococcal CovR Response Regulator Causes Dimerization and Promoter-Specific Recruitment by RNA Polymerase." Journal of Bacteriology 188, no. 13 (July 1, 2006): 4620–26. http://dx.doi.org/10.1128/jb.00198-06.

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ABSTRACT The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR.

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Sinha, Akesh, Sankalp Gupta, Shweta Bhutani, Anuj Pathak, and Dibyendu Sarkar. "PhoP-PhoP Interaction at Adjacent PhoP Binding Sites Is Influenced by Protein Phosphorylation." Journal of Bacteriology 190, no. 4 (December 7, 2007): 1317–28. http://dx.doi.org/10.1128/jb.01074-07.

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ABSTRACT Mycobacterium tuberculosis PhoP regulates the expression of unknown virulence determinants and the biosynthesis of complex lipids. PhoP, like other members of the OmpR family, comprises a phosphorylation domain at the amino-terminal half and a DNA-binding domain at the carboxy-terminal half of the protein. To explore structural effect of protein phosphorylation and to examine effect of phosphorylation on DNA binding, purified PhoP was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and Asp-71. Protein phosphorylation was not required for DNA binding; however, phosphorylation enhanced in vitro DNA binding through protein-protein interaction(s). Evidence is presented here that the protein-protein interface is different in the unphosphorylated and phosphorylated forms of PhoP and that specific DNA binding plays a critical role in changing the nature of the protein-protein interface. We show that phosphorylation switches the transactivation domain to a different conformation, which specifies additional protein-protein contacts between PhoP protomers bound to adjacent cognate sites. Together, our observations raise the possibility that PhoP, in the unphosphorylated and phosphorylated forms, may be capable of adopting different orientations as it binds to a vast array of genes to activate or repress transcription.

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Tierrez, Alberto, and Francisco García-del Portillo. "The Salmonella Membrane Protein IgaA Modulates the Activity of the RcsC-YojN-RcsB and PhoP-PhoQ Regulons." Journal of Bacteriology 186, no. 22 (November 15, 2004): 7481–89. http://dx.doi.org/10.1128/jb.186.22.7481-7489.2004.

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ABSTRACT The Salmonella enterica serovar Typhimurium membrane protein IgaA and the PhoP-PhoQ two-component system are used by this pathogen to attenuate the intracellular growth rate within fibroblasts. IgaA has also recently been shown to contribute to virulence by exerting tight repression of the RcsC-YojN-RcsB phosphorelay in host tissues. Here we show that loss of repression of the RcsC-YojN-RcsB system, linked to an R188H mutation in the IgaA protein (igaA1 allele), is accompanied by altered expression of PhoP-PhoQ-activated (pag) genes. The changes in gene expression were different depending on the specific pag gene analyzed. Thus, transcription of ugd, which is required for lipopolysaccharide modification and colanic acid capsule synthesis, was enhanced in the igaA1 mutant. RcsB and its coregulator RcsA promoted this alteration in a PhoP-PmrA-independent manner. Unlike ugd, activation of the RcsC-YojN-RcsB phosphorelay negatively affected the expression of all other pag genes tested. In this case, RcsB alone was responsible for this effect. We also found that PhoP, but not PmrA, negatively modulates the expression of gmm, a gene required for colanic acid synthesis that is regulated positively by RcsC-YojN-RcsB. Finally, it was observed that the fine regulation of pag genes exerted by RcsB requires the RpoS protein and that an active RcsB, but not RcsA, diminishes expression of the phoP gene. These data support the hypothesis that in Salmonella there is an intimate regulatory circuit between the PhoP-PhoQ and RcsC-YojN-RcsB phosphorelays, which is revealed only when the RcsC-YojN-RcsB signaling route is derepressed. Consistent with the phenotypes observed in fibroblast cells, IgaA is predicted to favor expression of the entire PhoP-PhoQ regulon based on its repression of the RcsC-YojN-RcsB phosphorelay.

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Bingle, Wade H., and John Smit. "Alkaline phosphatase and a cellulase reporter protein are not exported from the cytoplasm when fused to large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein." Canadian Journal of Microbiology 40, no. 9 (September 1, 1994): 777–82. http://dx.doi.org/10.1139/m94-122.

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Using a gene fusion approach, hybrid proteins were created by linking alkaline phosphatase (PhoA) or a cellulase reporter (ΔCenA) to four large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein (RsaA; 1026 amino acids). Three of the sites (amino acids 189, 220, 315) were selected on the basis of TnphoA experiments that suggested the first 250–350 amino acids of RsaA could mediate export of PhoA from the cytoplasm while the fourth lay only 21 amino acids from the C-terminus. Expression of all fusions except rsaA(315):ΔcenA and rsaA(315):phoA was toxic to C. crescentus. None of the gene fusions were toxic when expressed by Escherichia coli DH5α, where all the hybrid proteins accumulated as inclusion bodies. The toxicity of hybrid proteins encoding 189, 220, and 1005 RsaA-derived amino acids was related to the nature of the hybrid protein itself because truncated RsaA peptides lacking their reporter domains were nontoxic. Further study of RsaA(ΔC21) showed that this and presumably other truncated RsaA derivatives were neither secreted nor prone to intracellular accumulation. Although C. crescentus tolerated the expression of rsaA(3l5):ΔcenA and rsaA(315):phoA, the encoded hybrid proteins were not exported in significant quantities from the cytoplasm. These results extend and confirm earlier work that large portions of the S-layer protein N-terminus cannot mediate export of passenger proteins from the cytoplasm and that the entire native S-layer protein may be required to properly interact with the RsaA secretion machinery.Key words: surface layer, secretion, reporter protein, Caulobacter crescentus.

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Nagy, Zoltan, Shane Comer, and Albert Smolenski. "Analysis of Protein Phosphorylation Using Phos-Tag Gels." Current Protocols in Protein Science 93, no. 1 (July 25, 2018): e64. http://dx.doi.org/10.1002/cpps.64.

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Morris, R. Tyler, Espen E. Spangenburg, and Frank W. Booth. "Responsiveness of cell signaling pathways during the failed 15-day regrowth of aged skeletal muscle." Journal of Applied Physiology 96, no. 1 (January 2004): 398–404. http://dx.doi.org/10.1152/japplphysiol.00454.2003.

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Various cellular signaling pathways, such as phosphatidylinositol 3-kinase, calcineurin, Janus kinase 2/signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase (MAPK) have been suggested to play an important role in skeletal muscle growth. Old muscle, compared with young muscle, lacks the ability to completely regrow its muscle mass after an atrophy-induced stimulus. it is hypothesized that defects and/or delays in the activation of specific cell signaling pathways of aged soleus muscle limit the potential for growth. To test this, 42 male Fischer 344 × Brown Norway rats, 30 mo old, were hindlimb immobilized for 10 days, and their muscle samples were compared with muscle samples analyzed from 3- to 4-mo-old rats in a previous report (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391–C398, 2003). After 10 days, the immobilization was removed and rats were allowed to ambulate for a series of days. Alterations in the activation or deactivation status of specific signaling pathways were determined by comparing the phosphorylation (phos) and total concentration of specific signaling proteins (pan) through Western blotting with the 10-day immobilization group. Various cell signals and their respective time groups of the old rats were shown to be significantly different compared with the 10-day immobilization group. For example, peak increases during recovery from the immobilization were observed at 1) the third recovery day for calcineurin B-pan and 2) the sixth recovery day for glycogen synthase kinase-3β-phos, p70 S6 kinase (p70S6k) -phos and -pan, calcineurin A-pan, STAT3-phos and -pan, p44 MAPK-pan, and p42 MAPK-pan. In contrast, Akt-pan, c-Jun NH2-terminal kinase-phos, and p38 MAPK-phos were observed to decrease from 10-day immobilization values to control levels. Also, Aktphos was unchanged among all groups. In a follow-up experiment in which muscle samples from both the present study and a previous study (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391–C398, 2003) were reanalyzed together, the recovery-induced increase in p70S6k-phos from immobilization-atrophy was significantly attenuated in soleus muscles of the old group.

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Henry, Theresa C., Juliette E. Power, Christine L. Kerwin, Aishat Mohammed, Jonathan S. Weissman, Dale M. Cameron, and Dennis D. Wykoff. "Systematic Screen of Schizosaccharomyces pombe Deletion Collection Uncovers Parallel Evolution of the Phosphate Signal Transduction Pathway in Yeasts." Eukaryotic Cell 10, no. 2 (December 17, 2010): 198–206. http://dx.doi.org/10.1128/ec.00216-10.

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ABSTRACT The phosphate signal transduction (PHO) pathway, which regulates genes in response to phosphate starvation, is well defined in Saccharomyces cerevisiae . We asked whether the PHO pathway was the same in the distantly related fission yeast Schizosaccharomyces pombe . We screened a deletion collection for mutants aberrant in phosphatase activity, which is primarily a consequence of pho1 + transcription. We identified a novel zinc finger-containing protein (encoded by spbc27b12.11c + ), which we have named pho7 + , that is essential for pho1 + transcriptional induction during phosphate starvation. Few of the S. cerevisiae genes involved in the PHO pathway appear to be involved in the regulation of the phosphate starvation response in S. pombe . Only the most upstream genes in the PHO pathway in S. cerevisiae ( ADO1 , DDP1 , and PPN1 ) share a similar role in both yeasts. Because ADO1 and DDP1 regulate ATP and IP 7 levels, we hypothesize that the ancestor of these yeasts must have sensed similar metabolites in response to phosphate starvation but have evolved distinct mechanisms in parallel to sense these metabolites and induce phosphate starvation genes.

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Brazas, R. M., and D. J. Stillman. "The Swi5 zinc-finger and Grf10 homeodomain proteins bind DNA cooperatively at the yeast HO promoter." Proceedings of the National Academy of Sciences 90, no. 23 (December 1, 1993): 11237–41. http://dx.doi.org/10.1073/pnas.90.23.11237.

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SWI5 encodes a zinc-finger protein required for expression of the yeast HO gene. Using Swi5 protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the Swi5 stimulatory factor as the product of the GRF10 gene, which encodes a yeast homeodomain protein. GRF10, also known as PHO2 and BAS2, is a transcriptional activator of the PHO5 acid phosphatase gene and the HIS4 histidine biosynthesis gene. Grf10 protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disassociation rates indicates that the Grf10-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or Grf10. Finally, we show that HO expression is reduced in yeast strains containing grf10 null mutations and that full expression of a heterologous promoter containing a SWI5-dependent HO upstream activation sequence element requires GRF10.

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Rodriguez-Quinones, Francisco, Santiago Hernández-Allés, Sebastian Albertí, Pablo V. Escribá, and Vicente J. Benedí. "A novel plasmid series for in vitro production of phoA translational fusions and its use in the construction of Escherichia coli PhoE: :PhoA hybrid proteins." Gene 151, no. 1-2 (December 1994): 125–30. http://dx.doi.org/10.1016/0378-1119(94)90642-4.

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Możejko-Ciesielska, Justyna, and Luísa S. Serafim. "Proteomic Response of Pseudomonas putida KT2440 to Dual Carbon-Phosphorus Limitation during mcl-PHAs Synthesis." Biomolecules 9, no. 12 (November 28, 2019): 796. http://dx.doi.org/10.3390/biom9120796.

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Pseudomonas putida KT2440, one of the best characterized pseudomonads, is a metabolically versatile producer of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) that serves as a model bacterium for molecular studies. The synthesis of mcl-PHAs is of great interest due to their commercial potential. Carbon and phosphorus are the essential nutrients for growth and their limitation can trigger mcl-PHAs’ production in microorganisms. However, the specific molecular mechanisms that drive this synthesis in Pseudomonas species under unfavorable growth conditions remain poorly understood. Therefore, the proteomic responses of Pseudomonas putida KT2440 to the limited carbon and phosphorus levels in the different growth phases during mcl-PHAs synthesis were investigated. The data indicated that biopolymers’ production was associated with the cell growth of P. putida KT2440 under carbon- and phosphorus-limiting conditions. The protein expression pattern changed during mcl-PHAs synthesis and accumulation, and during the different physiological states of the microorganism. The data suggested that the majority of metabolic activities ceased under carbon and phosphorus limitation. The abundance of polyhydroxyalkanoate granule-associated protein (PhaF) involved in PHA synthesis increased significantly at 24 and 48 h of the cultivations. The activation of proteins belonging to the phosphate regulon was also detected. Moreover, these results indicated changes in the protein profiles related to amino acids metabolism, replication, transcription, translation, stress response mechanisms, transport or signal transduction. The presented data allowed the investigation of time-course proteome alterations in response to carbon and phosphorus limitation, and PHAs synthesis. This study provided information about proteins that can be potential targets in improving the efficiency of mcl-PHAs synthesis.

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Macfarlane, Emma L. A., Agnieszka Kwasnicka, Martina M. Ochs, and Robert E. W. Hancock. "PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance." Molecular Microbiology 34, no. 2 (October 1999): 305–16. http://dx.doi.org/10.1046/j.1365-2958.1999.01600.x.

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Eguchi, Y., J. Itou, M. Yamane, R. Demizu, F. Yamato, A. Okada, H. Mori, A. Kato, and R. Utsumi. "B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli." Proceedings of the National Academy of Sciences 104, no. 47 (November 12, 2007): 18712–17. http://dx.doi.org/10.1073/pnas.0705768104.

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Journal articles on the topic 'Proteine phos'

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