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.

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Available from: David G Drubin, Jan 09, 2014
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    • "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. "
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    • "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. "
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    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.
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