Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
The major facilitator superfamily (MFS) transporter Pho84 and the type III transporter Pho89 are responsible for metabolic effects of inorganic phosphate in yeast. While the Pho89 ortholog Pit1 was also shown to be involved in phosphate-activated MAPK in mammalian cells, it is currently unknown, whether orthologs of Pho84 have a role in phosphate-sensing in metazoan species. We show here that the activation of MAPK by phosphate observed in mammals is conserved in Drosophila cells, and used this assay to characterize the roles of putative phosphate transporters. Surprisingly, while we found that RNAi-mediated knockdown of the fly Pho89 ortholog dPit had little effect on the activation of MAPK in Drosophila S2R+ cells by phosphate, two Pho84/SLC17A1-9 MFS orthologs (MFS10 and MFS13) specifically inhibited this response. Further, using a Xenopus oocyte assay, we show that MSF13 mediates uptake of [(33)P]-orthophosphate in a sodium-dependent fashion. Consistent with a role in phosphate physiology, MSF13 is expressed highest in the Drosophila crop, midgut, Malpighian tubule, and hindgut. Altogether, our findings provide the first evidence that Pho84 orthologs mediate cellular effects of phosphate in metazoan cells. Finally, while phosphate is essential for Drosophila larval development, loss of MFS13 activity is compatible with viability indicating redundancy at the levels of the transporters.
???displayArticle.pubmedLink???
22359624 ???displayArticle.pmcLink???PMC3280997 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 1. Western blot analysis of phosphate-induced MAPK in S2R+ and Kc167 cells.A: Dose response and time course of phosphate-activated MAPK in S2R+ cells. B: Dose response of phosphate-activated MAPK in S2R+ cells. C: Time course of phosphate-activated MAPK in S2R+ cells. D: Time course of phosphate-activated MAPK in Kc167 cells. E: Effect of PFA on activation of MAPK in S2R+ cells Shown are one representative Western blot autoradiogram (A), or pooled densitometic data of at least three independent Western blot experiments (B–E). Abbreviations: Pi = inorganic phosphate, Ins = human insulin, Ly = Ly294002 (PI3K-inhibitor, 50 uM), PFA = phosphonoformic acid (30 mM, unless otherwise noted), S10 = sodium sulfate (10 mM), P10 = sodium phosphate (10 mM).
Figure 2. Blast and Bayes analysis of MFS transporters.Heatmap of pairwise BLAST bit scores for all known yeast, Drosophila and human proteins containing the MFS protein domain PF07690 [38] (left panel) sorted by a hierarchical clustering (middle panel). Bayesian phylogenetic reconstruction (dendogram) was used to identify 29 fly orthologs that are most closely related to yeast Pho84 (YML123C) and human SLC17A1–9. Posterior probabilities are indicated above each branch. Fly transporters found to be expressed in S2R+ cells are shown in bold/italic script.
Figure 3. Effect of RNAi knockdown of MFS transporters and dPit on MAPK.A: mRNA expression of MFS and Pit transporters in S2R+ cells. Data of three replicate experiments are shown as mean±SEM expression relative to actin 5 C. B: Effect of RNAi knockdown of MFS transporters and dPit on MAPK. Data of three replicate experiments are shown as mean±SEM relative to cells transfected with dsRNA targeting lucifierase (luc). C: RNAi knockdown efficiency. To calculate efficiency of knockdown, parallel wells prepared for pERK1/2 Western analysis above (Fig. 2B) were used for total RNA extraction and quantitative RT-PCR. Shown are mean±SEM of three replicate experiments after expression was corrected for actin 5 C mRNA. Cells treated with dsRNA targeting luciferase are set 100% for each specific primer pair.
Figure 4. Phosphate transport after expression of MFS and dPit transporters in X. oocytes.Phosphate uptake of Xenopus oocytes injected with capped RNA encoding MFS10 (FBgn0030452), MFS13 (FBgn0010497), and dPit (FBgn0260795), was measured in ND100+33P, or ND0+33P, in the presence or absence of 5 mM PFA at pH7.4 or at pH5.5 or 8.5 where indicated. 33P-uptake is expressed in multiples over basal seen with non-injected oocytes as mean±SEM from at least three different batches of oocytes (n = 10/bath).
Beck,
Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent.
2003, Pubmed
Beck,
Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent.
2003,
Pubmed Beck,
The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development.
2010,
Pubmed Beck,
Identification of a novel function of PiT1 critical for cell proliferation and independent of its phosphate transport activity.
2009,
Pubmed Bergwitz,
Phosphate sensing.
2011,
Pubmed Bergwitz,
Disorders of phosphate homeostasis and tissue mineralisation.
2009,
Pubmed Bun-Ya,
The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter.
1991,
Pubmed Celniker,
Unlocking the secrets of the genome.
2009,
Pubmed Chang,
Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells.
2006,
Pubmed Chertow,
A randomized trial of sevelamer hydrochloride (RenaGel) with and without supplemental calcium. Strategies for the control of hyperphosphatemia and hyperparathyroidism in hemodialysis patients.
1999,
Pubmed Chintapalli,
Using FlyAtlas to identify better Drosophila melanogaster models of human disease.
2007,
Pubmed Collins,
The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors.
2004,
Pubmed Festing,
Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1.
2009,
Pubmed Finn,
The Pfam protein families database.
2008,
Pubmed Friedman,
A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling.
2006,
Pubmed Gutiérrez,
Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis.
2008,
Pubmed Hsieh,
Global regulation by the seven-component Pi signaling system.
2010,
Pubmed Hubbard,
Ensembl 2009.
2009,
Pubmed Hubbard,
Ensembl 2007.
2007,
Pubmed Jaureguiberry,
A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc.
2008,
Pubmed
,
Xenbase Julien,
Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1.
2009,
Pubmed Julien,
Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway.
2007,
Pubmed Khoshniat,
The emergence of phosphate as a specific signaling molecule in bone and other cell types in mammals.
2011,
Pubmed Kulkarni,
Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays.
2006,
Pubmed Lamarche,
The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis.
2008,
Pubmed Mansfield,
Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism.
2001,
Pubmed Mizobuchi,
Vascular calcification: the killer of patients with chronic kidney disease.
2009,
Pubmed Morishita,
The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc.
2001,
Pubmed Mouillon,
New aspects on phosphate sensing and signalling in Saccharomyces cerevisiae.
2006,
Pubmed Mozar,
High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells.
2008,
Pubmed Murer,
The sodium phosphate cotransporter family SLC34.
2004,
Pubmed Nair,
Phosphocitrate inhibits a basic calcium phosphate and calcium pyrophosphate dihydrate crystal-induced mitogen-activated protein kinase cascade signal transduction pathway.
1997,
Pubmed Ohnishi,
Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging.
2010,
Pubmed Ponton,
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster.
2011,
Pubmed Razzaque,
Does FGF23 toxicity influence the outcome of chronic kidney disease?
2009,
Pubmed Reimer,
Organic anion transport is the primary function of the SLC17/type I phosphate transporter family.
2004,
Pubmed Ronquist,
MrBayes 3: Bayesian phylogenetic inference under mixed models.
2003,
Pubmed Saier,
The major facilitator superfamily.
1999,
Pubmed Segal,
Genetic transformation of Drosophila cells in culture by P element-mediated transposition.
1996,
Pubmed Sims,
FLIGHT: database and tools for the integration and cross-correlation of large-scale RNAi phenotypic datasets.
2006,
Pubmed Sprecher,
Familial tumoral calcinosis: from characterization of a rare phenotype to the pathogenesis of ectopic calcification.
2010,
Pubmed Stubbs,
Role of hyperphosphatemia and 1,25-dihydroxyvitamin D in vascular calcification and mortality in fibroblastic growth factor 23 null mice.
2007,
Pubmed Suzuki,
Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism.
2010,
Pubmed Tenenhouse,
Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidney.
1989,
Pubmed Toh-e,
PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28, of Saccharomyces cerevisiae.
1988,
Pubmed Werner,
Evolution of the Na-P(i) cotransport systems.
2001,
Pubmed
,
Xenbase Wittrant,
Inorganic phosphate regulates Glvr-1 and -2 expression: role of calcium and ERK1/2.
2009,
Pubmed Yamazaki,
Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells.
2010,
Pubmed Yanagawa,
Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling.
1998,
Pubmed Yoshiko,
Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization.
2007,
Pubmed
