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: 4.47). 04/2012; 23(12):2388-98. DOI: 10.1091/mbc.E12-03-0209
Source: PubMed


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.

Download full-text


Available from: David G Drubin, Jan 09, 2014
63 Reads
  • Source
    • "Extraction and processing of LCBs from yeast cells for fluorescence high-performance liquid chromatography (HPLC) analysis using the AQC reagent (Waters, Milford, MS) were performed as described previously (Lester and Dickson 2001; Sun et al. 2012). Briefly, HPLC analysis was performed using a C18 column (4.6 9 250 mm, XDB-C18; Hewlett-Packard, Palo Alto, CA) on a Shimadzu LC10A series liquid chromatography system. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sphingolipids are a family of eukaryotic lipids biosynthesized from sphingoid long-chain bases (LCBs). Sphingolipids are an essential class of lipids, as their depletion results in cell death. However, acute LCB supplementation is also toxic; thus, proper cellular LCB levels should be maintained. To characterize the "sphingolipid-signaling intercross," we performed a kinome screening assay in which budding yeast protein kinase-knockout strains were screened for resistance to ISP-1, a potent inhibitor of LCB biosynthesis. Here, one pair of such DIR (deletion-mediated ISP-1 resistance) genes, FPK1 and FPK2, was further characterized. Cellular LCB levels increased in the fpk1/2∆ strain, which was hypersensitive to phytosphingosine (PHS), a major LCB species of yeast cells. Concomitantly, this strain acquired resistance to ISP-1. Fpk1 and Fpk2 were involved in two downstream events; that is, ISP-1 uptake due to aminophospholipid flippase and LCB degradation due to LCB4 expression. RSK3, which belongs to the p90-S6K subfamily, was identified as a functional counterpart of Fpk1/2 in mammalian cells as the RSK3 gene functionally complemented the ISP-1-resistant phenotype of fpk1/2∆ cells.
    MicrobiologyOpen 04/2014; 3(2). DOI:10.1002/mbo3.160 · 2.21 Impact Factor
  • Source
    • "of which play integral roles in the pathway (Figure 3A). No interaction was observed with TORC1, in good agreement with prior findings (Roelants et al., 2011; Sun et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Current approaches for identifying synergistic targets use cell culture models to see if the combined effect of clinically available drugs is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that may be synergistic targets. Here, we created a tool to screen and identify molecular targets that may synergize with new inhibitors of target of rapamycin (TOR), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. Although clinical results from TOR complex 1 (TORC1)-specific inhibition using rapamycin analogs have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand this increased therapeutic efficacy and to discover secondary targets for combination therapy, we engineered Tor2 in S. cerevisiae to accept an orthogonal inhibitor. We used this tool to create a chemical epistasis miniarray profile (ChE-MAP) by measuring interactions between the chemically inhibited Tor2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1- and TORC2-dependent functions. The results showed a TORC2-specific interaction with the pentose phosphate pathway, a previously unappreciated TORC2 function that suggests a role for the complex in balancing the high energy demand required for ribosome biogenesis.
    Cell Reports 12/2013; 5(6). DOI:10.1016/j.celrep.2013.11.040 · 8.36 Impact Factor
  • Source
    • "In S. cerevisiae, Ypk1 activates the first enzyme in the sphingolipid biosynthesis pathway through the control of the Orm protein activity [21]. Recent studies reported that Ypk1 regulates the sphingolipid homeostasis during heat stress through Orm protein phosphorylation [62]. A similar mechanism may occur in A. nidulans, as low levels of YpkA increased the susceptibility of the cells to restrictive temperatures, probably due to the absence of key elements of the cellular structure. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The and genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the homologue, YpkA, representing the first filamentous fungal homologue. Two conditional mutant strains were constructed by replacing the endogenous promoter with two different regulatable promoters, (from the alcohol dehydrogenase gene) and (from the nitrate reductase gene). Both constructs confirmed that was an essential gene in . Repression of caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The Pkh1 homologue was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of or epistatic to , rather, and are genetically independent or in parallel. is a homologue of the yeast acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When was absent, repression was lethal to the cell. Therefore, there appears to be a genetic interaction between , , and the sphingolipid synthesis. Transcriptional profiling of overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.
    PLoS ONE 03/2013; 8(3):e57630. DOI:10.1371/journal.pone.0057630 · 3.23 Impact Factor
Show more