Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Molecular biology of the cell (Impact Factor: 5.98). 04/2012; 23(12):2388-98. DOI: 10.1091/mbc.E12-03-0209
Source: PubMed

ABSTRACT Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55-protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Target Of Rapamycin (TOR) is a Ser/Thr kinase that operates in two conserved, multiprotein complexes, TORC1 and TORC2. Unlike TORC1, TORC2 is insensitive to rapamycin and its functional characterization is less advanced. Previous genetic studies demonstrated that TORC2 depletion leads to loss of actin polarization and endocytosis. To determine how TORC2 regulates these readouts, we engineered a yeast strain in which TORC2 can be specifically and acutely inhibited by the imidazoquinoline NVP-BHS345. Kinetic analyses following inhibition of TORC2, supported with quantitative phosphoproteomics revealed that TORC2 regulates these readouts via distinct pathways: rapidly through direct protein phosphorylation cascades and slowly through indirect changes in the tensile properties of the plasma membrane. The rapid signaling events are mediated in large part through the phospholipid flippase kinases Fpk1 and Fpk2 while the slow signaling pathway involves increased plasma membrane tension resulting from a gradual depletion of sphingolipids. Additional hits in our phosphoproteomics screens highlight the intricate control TORC2 exerts over diverse aspects of eukaryote cell physiology. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 04/2015; DOI:10.1074/jbc.M114.627794 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In broilers, high ambient temperature can result in reduced feed consumption, digestive inefficiency, impaired metabolism, and even death. The broiler sector of the U.S. poultry industry incurs approximately $52 million in heat-related losses annually. The objective of this study is to characterize the effects of cyclic high ambient temperature on the transcriptome of a metabolically active organ, the liver. This study provides novel insight into the effects of high ambient temperature on metabolism in broilers, because it is the first reported RNA-seq study to characterize the effect of heat on the transcriptome of a metabolic-related tissue. This information provides a platform for future investigations to further elucidate physiologic responses to high ambient temperature and seek methods to ameliorate the negative impacts of heat. Transcriptome sequencing of the livers of 8 broiler males using Illumina HiSeq 2000 technology resulted in 138 million, 100-base pair single end reads, yielding a total of 13.8 gigabases of sequence. Forty genes were differentially expressed at a significance level of P-value < 0.05 and a fold-change >= 2 in response to a week of cyclic high ambient temperature with 27 down-regulated and 13 up-regulated genes. Two gene networks were created from the function-based Ingenuity Pathway Analysis (IPA) of the differentially expressed genes: "Cell Signaling" and "Endocrine System Development and Function". The gene expression differences in the liver transcriptome of the heat-exposed broilers reflected physiological responses to decrease internal temperature, reduce hyperthermia-induced apoptosis, and promote tissue repair. Additionally, the differential gene expression revealed a physiological response to regulate the perturbed cellular calcium levels that can result from high ambient temperature exposure. Exposure to cyclic high ambient temperature results in changes at the metabolic, physiologic, and cellular level that can be characterized through RNA-seq analysis of the liver transcriptome of broilers. The findings highlight specific physiologic mechanisms by which broilers reduce the effects of exposure to high ambient temperature. This information provides a foundation for future investigations into the gene networks involved in the broiler stress response and for development of strategies to ameliorate the negative impacts of heat on animal production and welfare.
    BMC Genomics 12/2014; 15(1):1084. DOI:10.1186/1471-2164-15-1084 · 4.04 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known. We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice. Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice. Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
    The Journal of allergy and clinical immunology 04/2015; DOI:10.1016/j.jaci.2015.02.031 · 11.25 Impact Factor