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1.
J Biol Chem ; 295(7): 2043-2056, 2020 02 14.
Article in English | MEDLINE | ID: mdl-31848224

ABSTRACT

The environmental stress response (ESR) is critical for cell survival. Yeast cells unable to synthesize inositol pyrophosphates (PP-InsPs) are unable to induce the ESR. We recently discovered a diphosphoinositol pentakisphosphate (PP-InsP5) phosphatase in Saccharomyces cerevisiae encoded by SIW14 Yeast strains deleted for SIW14 have increased levels of PP-InsPs. We hypothesized that strains with high inositol pyrophosphate levels will have an increased stress response. We examined the response of the siw14Δ mutant to heat shock, nutrient limitation, osmotic stress, and oxidative treatment using cell growth assays and found increased resistance to each. Transcriptional responses to oxidative and osmotic stresses were assessed using microarray and reverse transcriptase quantitative PCR. The ESR was partially induced in the siw14Δ mutant strain, consistent with the increased stress resistance, and the mutant strain further induced the ESR in response to oxidative and osmotic stresses. The levels of PP-InsPs increased in WT cells under oxidative stress but not under hyperosmotic stress, and they were high and unchanging in the mutant. Phosphatase activity of Siw14 was inhibited by oxidation that was reversible. To determine how altered PP-InsP levels affect the ESR, we performed epistasis experiments with mutations in rpd3 and msn2/4 combined with siw14Δ. We show that mutations in msn2Δ and msn4Δ, but not rpd3, are epistatic to siw14Δ by assessing growth under oxidative stress conditions and expression of CTT1 Msn2-GFP nuclear localization was increased in the siw14Δ. These data support a model in which the modulation of PP-InsPs influence the ESR through general stress response transcription factors Msn2/4.


Subject(s)
DNA-Binding Proteins/genetics , Oxidative Stress/genetics , Protein Tyrosine Phosphatases/genetics , Saccharomyces cerevisiae Proteins/genetics , Stress, Physiological/genetics , Transcription Factors/genetics , Cell Cycle/genetics , Cell Survival/genetics , DNA-Binding Proteins/metabolism , Diphosphates/metabolism , Gene Expression Regulation, Fungal/genetics , Inositol/metabolism , Osmotic Pressure/drug effects , Oxidation-Reduction , Peptides, Cyclic/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/genetics , Transcription Factors/metabolism
2.
J Biol Chem ; 294(12): 4621-4633, 2019 03 22.
Article in English | MEDLINE | ID: mdl-30659094

ABSTRACT

Phosphoinositide 3-kinase ß (PI3Kß) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110ß) of PI3Kß whose mutation disrupts binding to Rab5. To better define the Rab5-p110ß interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110ß helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gßγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110ß as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kß interaction by hydrogen-deuterium exchange MS identified p110ß peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110ß or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110ß had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kß activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110ß helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kß coupling in vivo.


Subject(s)
Breast Neoplasms/pathology , Neoplasm Invasiveness , Phosphatidylinositol 3-Kinase/metabolism , rab5 GTP-Binding Proteins/metabolism , Binding Sites , Breast Neoplasms/metabolism , Cell Line, Tumor , Chemotaxis , Gelatin/metabolism , HEK293 Cells , Humans , Mass Spectrometry/methods , Mutation , Phosphatidylinositol 3-Kinase/genetics , Protein Binding
3.
J Biol Chem ; 291(13): 6772-83, 2016 Mar 25.
Article in English | MEDLINE | ID: mdl-26828065

ABSTRACT

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the ß-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5or IP7)in vitro. In vivo,siw14Δ yeast mutants possess increased IP7levels, whereas heterologousSIW14overexpression eliminates IP7from cells. IP7levels increased proportionately whensiw14Δ was combined withddp1Δ orvip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7isoform 5PP-IP5to IP6.


Subject(s)
Gene Expression Regulation, Fungal , Inositol Phosphates/metabolism , Protein Tyrosine Phosphatases/metabolism , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Deletion , Genetic Complementation Test , Kinetics , Phosphotransferases (Phosphate Group Acceptor)/genetics , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Protein Tyrosine Phosphatases/genetics , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Substrate Specificity
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