Phosphate Signal Transduction Pathway Research Articles

Cyclin-dependent kinase Pho85p and its cyclins are involved in replicative lifespan through multiple pathways in yeast.

Lifespan is determined by genetic factors and influenced by environmental factors. Here, we find that the phosphate signal transduction (PHO) pathway is involved in the determination of replicative lifespan in budding yeast. Extracellular phosphate does not affect the lifespan. However, deletion of PHO80 (cyclin) and PHO85 (cyclin-dependent kinase) genes, that is, negative regulators of the PHO pathway, shortens the lifespan, which is restored by further deletion of PHO4 (transcriptional activator). Four of the other nine Pho85p cyclin genes are also required to maintain normal lifespan. The short-lived mutants show a metabolic profile that is similar to strains with normal lifespan. Thus, Pho85p kinase genetically determines replicative lifespan in combination with relevant cyclins. Our findings uncover novel cellular signals in longevity regulation.

Phosphate Assay Kit in One Cell for Electrochemical Detection of Intracellular Phosphate Ions at Single Cells

In this paper, phosphate assay kit in one cell is realized for the electrochemical detection of intracellular phosphate ions at single cells. The components of the phosphate assay kit, including maltose phosphorylase, maltose, mutarotase, and glucose oxidase, are electrochemically injected into a living cell through a nanometer-sized capillary with the ring electrode at the tip. These components react with phosphate ions inside the cell to generate hydrogen peroxide that is electrochemically oxidized at the ring electrode for the qualification of intracellular phosphate ions. An average 1.7 nA charge was collected from eight individual cells, suggesting an intracellular phosphate concentration of 2.1 mM. The establishment in the electrochemical measurement of phosphate ions provides a special strategy to monitor the fluctuation of intracellular phosphate at single cells, which is significant for the future investigation of phosphate signal transduction pathway.

Core regulatory components of the PHO pathway are conserved in the methylotrophic yeast Hansenula polymorpha.

To gain better understanding of the diversity and evolution of the gene regulation system in eukaryotes, the phosphate signal transduction (PHO) pathway in non-conventional yeasts has been studied in recent years. Here we characterized the PHO pathway of Hansenula polymorpha, which is genetically tractable and distantly related to Saccharomyces cerevisiae and Schizosaccharomyces pombe, in order to get more information for the diversity and evolution of the PHO pathway in yeasts. We generated several pho gene-deficient mutants based on the annotated draft genome of H. polymorpha BY4329. Except for the Hppho2-deficient mutant, these mutants exhibited the same phenotype of repressible acid phosphatase (APase) production as their S. cerevisiae counterparts. Subsequently, Hppho80 and Hppho85 mutants were isolated as suppressors of the Hppho81 mutation and Hppho4 was isolated from Hppho80 and Hppho85 mutants as the sole suppressor of the Hppho80 and Hppho85 mutations. To gain more complete delineation of the PHO pathway in H. polymorpha, we screened for UV-irradiated mutants that expressed APase constitutively. As a result, three classes of recessive constitutive mutations and one dominant constitutive mutation were isolated. Genetic analysis showed that one group of recessive constitutive mutations was allelic to HpPHO80 and that the dominant mutation occurred in the HpPHO81 gene. Epistasis analysis between Hppho81 and the other two classes of recessive constitutive mutations suggested that the corresponding new genes, named PHO51 and PHO53, function upstream of HpPHO81 in the PHO pathway. Taking these findings together, we conclude that the main components of the PHO pathway identified in S. cerevisiae are conserved in the methylotrophic yeast H. polymorpha, even though these organisms separated from each other before duplication of the whole genome. This finding is useful information for the study of evolution of the PHO regulatory system in yeasts.Electronic supplementary materialThe online version of this article (doi:10.1007/s00294-016-0565-7) contains supplementary material, which is available to authorized users.

A novel protein, Pho92, has a conserved YTH domain and regulates phosphate metabolism by decreasing the mRNA stability of PHO4 in Saccharomyces cerevisiae

The homologue of human YTHDF2, Ydr374c (Pho92), is the only protein that has a YTH (YT521-B homology) domain in Saccharomyces cerevisiae. Based on microarray analysis, genes involved in the phosphate signal transduction (PHO) pathway were up-regulated in the Δpho92 strain, as were genes regulated by Pho4, which is an important transcription factor in the PHO pathway. To identify the exact mechanism of Pho92 action with respect to phosphate metabolism, we investigated the effect of Pho92 on PHO4 expression. The half-life of PHO4 mRNA was increased in the Δpho92 strain; this phenotype was also observed in the deletion mutants UPF1 and POP2, which are components of the NMD (nonsense-mediated decay) pathway and the Pop2-Ccr4-Not deadenylase complex respectively. Pho92 interacts physically with Pop2 of the Pop2-Ccr4-Not deadenylase complex. Furthermore, Pho92 binding to the 3'-UTR of PHO4 was dependent on the phosphate concentration. Deletion of the PHO4 3'-UTR resulted in PHO4 mRNA resistance to Pho92-dependent degradation. The results of the present study indicate that Pho92 regulates Pho4 expression at the post-transcriptional level via the regulation of mRNA stability. Taken together, Pho92 participates in cellular phosphate metabolism, specifically via the regulation of PHO4 mRNA stability by binding to the 3'-UTR in a phosphate-dependent manner.

Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe

BackgroundInorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway, leading to expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway involving genetically identified positive (pho7+) and negative (csk1+) regulators. The genes induced by phosphate limitation and the molecular mechanism by which pho7+ and csk1+ function are unknown. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response.ResultsWe define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response that contains the ScPHO5 (pho1+), ScPHO84 (SPBC8E4.01c), and ScGIT1 (SPBC1271.09) orthologs. We identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction.ConclusionsWe provide a global analysis of the transcriptional response to phosphate limitation in S. pombe. Our results elucidate the conserved core regulon induced in response to phosphate starvation in this ascomycete distantly related to S. cerevisiae and provide a better understanding of flexibility in environmental stress response networks.

Systematic Screen of Schizosaccharomyces pombe Deletion Collection Uncovers Parallel Evolution of the Phosphate Signal Transduction Pathway in Yeasts

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.

Novel Acid Phosphatase in Candida glabrata Suggests Selective Pressure and Niche Specialization in the Phosphate Signal Transduction Pathway

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

The SPX domain of the yeast low‐affinity phosphate transporter Pho90 regulates transport activity

Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2.

Candida glabrata PHO4 Is Necessary and Sufficient for Pho2-Independent Transcription of Phosphate Starvation Genes

Comparative genomic analyses of Candida glabrata and Saccharomyces cerevisiae suggest many signal transduction pathways are highly conserved. Focusing on the phosphate signal transduction (PHO) pathway of C. glabrata, we demonstrate that components of the pathway are conserved and confirm the role of CgPHO81, CgPHO80, CgPHO4, and CgMSN5 in the PHO pathway through deletion analysis. Unlike S. cerevisiae, C. glabrata shows little dependence on the transcription factor, Pho2, for induction of phosphate-regulated genes during phosphate limitation. We show that the CgPho4 protein is necessary and sufficient for Pho2-independent gene expression; CgPho4 is capable of driving expression of PHO promoters in S. cerevisiae in the absence of ScPHO2. On the basis of the sequences of PHO4 in the hemiascomycetes and complementation analysis, we suggest that Pho2 dependence is a trait only observed in species closely related to S. cerevisiae. Our data are consistent with trans-regulatory changes in the PHO pathway via the transcription factor Pho4 as opposed to cis-regulatory changes (the promoter).

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.

Intracellular Phosphate Serves as a Signal for the Regulation of the PHO Pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) is known to regulate the expression of several phosphate-responsive genes, such as PHO5 and PHO84. However, the fundamental issue of whether cells sense intracellular or extracellular phosphate remains unresolved. To address this issue, we have directly measured intracellular phosphate concentrations by (31)P NMR spectroscopy. We find that PHO5 expression is strongly correlated with the levels of both intracellular orthophosphate and intracellular polyphosphate and that the signaling defect in the Deltapho84 strain is likely to result from insufficient intracellular phosphate caused by a defect in phosphate uptake. Furthermore, the Deltaphm1Deltaphm2, Deltaphm3, and Deltaphm4 strains, which lack intracellular polyphosphate, have higher intracellular orthophosphate levels and lower expression of PHO5 than the wild-type strain. By contrast, the Deltaphm5 strain, which has lower intracellular orthophosphate and higher polyphosphate levels than the wild-type strain, shows repressed expression of PHO5, similar to the wild-type strain. These observations suggest that PHO5 expression is under the regulation of intracellular orthophosphate, although orthophosphate is not the sole signaling molecule. Moreover, the disruption of PHM3, PHM4, or of both PHM1 and PHM2 in the Deltapho84 strain suppresses, although not completely, the PHO5 constitutive phenotype by increasing intracellular orthophosphate, suggesting that Pho84p affects phosphate signaling largely by functioning as a transporter.

Priming effects of lipopolysaccharide on UTP-induced arachidonic acid release in RAW 264.7 macrophages

Stimulation of mouse RAW 264.7 macrophages with UTP activates both the inositol phosphate signal transduction pathway and the phospholipase A 2 pathway. In the present study, we investigated the interactions between bacterial lipopolysaccharide and UTP in these two systems and the underlying mechanisms involved. While the UTP-induced release of arachidonic acid was only 2.9-fold that in controls, priming the cells with 1 μg/ml lipopolysaccharide for 1 h before UTP treatment resulted in 9.2-fold arachidonic acid release upon stimulation with UTP. Lipopolysaccharide priming was both concentration- and time-dependent with a peak effect after 1 h treatment at a concentration of 1 μg/ml. Lipopolysaccharide treatment affected neither the basal nor the UTP-stimulated inositol phosphate formation and [Ca 2+] i rise. Pretreatment of the cells with staurosporine, calphostin, N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-7), genistein or K-252a led to marked inhibition of the priming effect, suggesting that both protein kinase C and tyrosine kinase are involved in the lipopolysaccharide effect. Buffering intracellular Ca 2+ levels using [1,2-bis-( o-aminophenoxyl)ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester] (BAPTA/AM) or pretreatment with either N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), 2-(2-amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one (PD098059) or {1-[ N,O-bis-(5-isoquinolinesulfonyl)- N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62) did not affect the lipopolysaccharide-induced priming effect. Primed UTP stimulation was inhibited by actinomycin D and cycloheximide, indicating a requirement for both gene expression and protein translation. To further examine whether the stimulatory effects of lipopolysaccharide on phospholipase A 2 activity were independent of [Ca 2+] i levels but dependent on protein phosphorylation, a fixed Ca 2+ concentration and inhibitors of protein phosphatases were used in primed permeabilized cells. Arachidonic acid release from permeabilized cells containing 100 nM Ca 2+ was high in lipopolysaccharide-primed cells and potentiated by addition of microcystin, orthovanadate or FK 506. These results suggest that the Ser/Thr and tyrosine phosphorylation cascades induced by protein kinase C and tyrosine kinase, respectively, are required for the arachidonic acid potentiation effect of lipopolysaccharide, which was independent of modulation of the upper stream signaling pathways of UTP.

Inhibition by cyclic AMP of basal and induced inositol phosphate production in cultured aortic smooth muscle cells from Wistar- Kyoto and spontaneously hypertensive rats

To investigate inositol phosphate formation and its modulation by the cyclic AMP (cAMP) pathway in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR). Phenylephrine was used to stimulate inositol phosphate formation in cultured aortic smooth muscle cells from SHR and Wistar-Kyoto (WKY) rats. The smooth muscle cells from passages 6-14 were prelabelled with myo-[2-3H]-inositol (1.9 x 10(5) Bq/ml for 24 h) and inositol phosphate formation was measured after exposure to agonist for 45 min. (-)isoproterenol or forskolin-induced cAMP formation was also evaluated using a radioimmunoassay method. The basal level of inositol phosphate formation in smooth muscle cells from SHR was higher than that observed in smooth muscle cells from WKY rats. Phenylephrine increased the formation of inositol phosphates in a concentration-dependent manner (0.1-100 mumol/l). In the presence of 100 mumol/l phenylephrine, the increase in inositol phosphate formation was significantly greater in smooth muscle cells from SHR (214 +/- 6%) than that observed in smooth muscle cells from WKY rats (156 +/- 8%). When the cells were pretreated with 1 mmol/l 8-bromoadenosine 3':5'-cyclic monophosphate or with 10 mumol/l forskolin for 45 min, the basal production of inositol phosphates in smooth muscle cells both from SHR and from WKY rats was significantly and similarly decreased by about 20%. In the presence of 1 mmol/l 8-bromoadenosine 3':5'-cyclic monophosphate, 100 mumol/l phenylephrine-induced inositol phosphate formation was similarly decreased by 33 +/- 4 and 30 +/- 3% in smooth muscle cells from SHR and from WKY rats, respectively, whereas, in the presence of 10 mumol/l forskolin, inositol phosphate formation was reduced by 25 +/- 3 and 27 +/- 5%, respectively, in those cells. In contrast, isoproterenol induced less inhibition of phenylephrine-induced inositol phosphate formation in smooth muscle cells from SHR (14 +/- 2%) than it did in those from WKY rats (25 +/- 4.5%). Although there was no significant difference in basal or forskolin-induced cAMP accumulation between smooth muscle cells from SHR and those from WKY rats. (-)isoproterenol-induced cAMP accumulation was significantly lower in smooth muscle cells from SHR. A marked inhibitory effect of cAMP on the alpha 1-adrenoceptor-mediated inositol phosphate signal transduction pathway was demonstrated in smooth muscle cells of SHR and of WKY rats. Decreased cAMP formation with beta-adrenergic stimulation and increased inositol phosphate formation with alpha-adrenergic stimulation in SHR smooth muscle cells may both contribute to the dominant alpha 1-adrenergic activity observed in SHR.

Selection and Characterization of Mammalian Cell Lines with Stable Over‐Expression of Human Pituitary Receptors for Gonadoliberin

The cDNA encoding the receptor for gonadoliberin (GnRH or LH-RH) was isolated from a human pituitary cDNA library and heterologously expressed in the murine fibroblast cell line LTK-. By using a dicistronic expression strategy utilizing the internal ribosomal-entry-site sequence of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturation-binding experiments, the gonadoliberin antagonist Cetrorelix showed high-affinity binding to the heterologously expressed human gonadoliberin receptor with a Kd of 0.1 nM. The pharmacological profile using 125I-Cetrorelix as radioligand and the authentic gonadoliberin or agonistic and antagonistic derivatives as competitors, showed a distinct rank order of binding potencies. Superagonistic gonadoliberin derivatives had more than ten-times higher binding affinities in comparison to gonadoliberin with a Kd of 3.47 nM. The gonadoliberin receptor expressed in stably transfected LTK- cells coupled to the inositol phosphate signal-transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5-trisphosphate in a dose-dependent way with an EC50 of 5 nM. This stimulatory effect of gonadoliberin was completely antagonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth-arrested cells, a transient expression of the c-fos protooncogene was induced by gonadoliberin or [D-Trp6]gonadoliberin, showing that the gonadoliberin receptor couples to a putative mitogenic signal-transduction pathway in this heterologous cell system.

Importance of arachidonic acid metabolites in regulating ATP-induced calcium fluxes in thyroid FRTL-5 cells

Stimulating rat thyroid FRTL-5 cells with the purinergic agonist ATP activates both the inositol phosphate signal-transduction pathway and the phospholipase A 2 pathway. In the present study we wanted to investigate the possible inter-relationships between these two systems during ATP-induced changes in intracellular free calcium ([Ca 2+] i). Pretreatment of Fura-2 loaded cells with 4-bromophenylacyl, an inhibitor of phospholipase A 2, had no effect on the ATP-induced entry of Ca 2+ but inhibited the release of sequestered Ca 2+. Nordihydrogualaretic acid (NDGA), a lipoxygenase inhibitor, and 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of cytochrome P-450 enzymes, attenuated the ATP-evoked transient increase in [Ca 2+] i. Furthermore, the capacitative entry of Ca 2+ was also attenuated in NDGA- and ETYA-treated cells stimulated with ATP. Similar results were obtained using econazole, an inhibitor of cytochrome P-450 enzymes. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the ATP-evoked response in [Ca 2+] i. We also showed that stimulation of intact or permeabilized FRTL-5 cells with arachidonic acid released sequestered calcium. This calcium originated, at least in part, from an IP 3 sensitive calcium pool. In addition, arachidonic acid rapidly acidified the cytosol. The results suggest that metabolism of arachidonic acid by a non-cyclooxygenase pathway is of importance in supporting agonist-induced calcium fluxes evoked via stimulation of the inositol phosphate pathway in FRTL-5 cells. Furthermore, arachidonic acid per se may modify agonist-induced calcium fluxes in these cells.