title: Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae.
abstract: The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine:palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1 cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1(ts) ypk2 cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1(T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.
authors.affiliation: Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Françoise M
authors.initials: FM
authors.lastname: Roelants
authors.forename: David K
authors.initials: DK
authors.lastname: Breslow
authors.forename: Alexander
authors.initials: A
authors.lastname: Muir
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism*; D015256:Coenzyme A-Transferases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006706:Homeostasis / Q000502:physiology*; D047468:Immunoprecipitation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism*; D013696:Temperature
chemical_list: D018797:Cell Cycle Proteins; D015256:Coenzyme A-Transferases; D005229:Fatty Acids, Monounsaturated; D038362:Glycogen Synthase Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; C548539:Orm1 protein, S cerevisiae; C548540:Orm2 protein, S cerevisiae; C081135:TOR2 protein, S cerevisiae; C068124:MCK1 protein, S cerevisiae; C001996:thermozymocidin
references.citation: Microbiology (Reading). 2004 Oct;150(Pt 10):3289-304
references.pmid: 15470109
references.citation: Cell. 2006 Oct 6;127(1):125-37
references.pmid: 16962653
references.citation: Nat Rev Mol Cell Biol. 2010 Jan;11(1):9-22
references.pmid: 20027184
references.citation: Curr Biol. 1999 Feb 25;9(4):186-97
references.pmid: 10074427
references.citation: Genetics. 2002 Aug;161(4):1453-64
references.pmid: 12196392
references.citation: Trends Cell Biol. 2001 Apr;11(4):167-72
references.pmid: 11306297
references.citation: Cell. 2010 Nov 12;143(4):639-50
references.pmid: 21035178
references.citation: Mol Cell Biol. 2000 Jun;20(12):4411-9
references.pmid: 10825204
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5701-2
references.pmid: 20304791
references.citation: Mol Cell Biol. 2011 Feb;31(4):721-35
references.pmid: 21135117
references.citation: Crit Rev Biochem Mol Biol. 2009 Sep-Oct;44(5):264-77
references.pmid: 19780638
references.citation: Biochem Biophys Res Commun. 1995 Jun 15;211(2):396-403
references.pmid: 7794249
references.citation: J Biochem. 2011 Feb;149(2):131-43
references.pmid: 21134888
references.citation: Sci Signal. 2009 Apr 21;2(67):pe24
references.pmid: 19383975
references.citation: J Biol Chem. 1998 Nov 13;273(46):30688-94
references.pmid: 9804843
references.citation: Arch Biochem Biophys. 2005 May 1;437(1):29-41
references.pmid: 15820214
references.citation: Nat Rev Mol Cell Biol. 2010 Aug;11(8):593-8
references.pmid: 20606693
references.citation: Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7899-902
references.pmid: 8058731
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 5;107(1):34-9
references.pmid: 19966303
references.citation: Genetics. 2011 Dec;189(4):1177-201
references.pmid: 22174183
references.citation: Nature. 2010 Feb 25;463(7284):1048-53
references.pmid: 20182505
references.citation: Mol Cell Biol. 2002 Mar;22(5):1329-39
references.pmid: 11839800
references.citation: Cell Metab. 2008 Feb;7(2):148-58
references.pmid: 18249174
references.citation: Curr Biol. 2009 Dec 1;19(22):R1046-52
references.pmid: 19948145
references.citation: Mol Cell Biol. 2005 Aug;25(16):7239-48
references.pmid: 16055732
references.citation: Mol Biol Cell. 2002 Sep;13(9):3005-28
references.pmid: 12221112
references.citation: Mol Biol Cell. 2008 Apr;19(4):1783-97
references.pmid: 18199685
references.citation: Nat Protoc. 2009;4(10):1513-21
references.pmid: 19798084
references.citation: Nature. 2002 Jul 25;418(6896):387-91
references.pmid: 12140549
references.citation: Trends Cell Biol. 2004 Dec;14(12):670-7
references.pmid: 15564043
references.citation: Mol Genet Genomics. 2010 Feb;283(2):111-22
references.pmid: 19967545
references.citation: J Lipid Res. 2008 May;49(5):909-21
references.pmid: 18296751
references.citation: J Cell Biol. 2009 Jun 29;185(7):1227-42
references.pmid: 19564405
references.citation: Cell Cycle. 2011 Jul 15;10(14):2305-16
references.pmid: 21670596
references.citation: J Cell Biol. 2002 Jan 21;156(2):241-8
references.pmid: 11807089