A protein kinase network regulates the function of aminophospholipid flippases

Limited exposure of aminophospholipids on the outer leaflet of the plasma membrane is a fundamental feature of eukaryotic cells and is maintained by the action of inward-directed P-type ATPases (“flippases”). Yeast S. cerevisiae has five flippases (Dnf1, Dnf2, Dnf3, Drs2, and Neo1), but their regulation is poorly understood. Two paralogous plasma membrane-associated protein kinases, Pkh1 and Pkh2 (orthologs of mammalian PDK1), are required for viability of S. cerevisiae cells because they activate several essential downstream protein kinases by phosphorylating a critical Thr in their activation loops. Two such targets are related protein kinases Ypk1 and Ypk2 (orthologs of mammalian SGK1), which have been implicated in multiple processes, including endocytosis and coupling of membrane expansion to cell wall remodeling, but the downstream effector(s) of these kinases have been elusive. Here we show that a physiologically relevant substrate of Ypk1 is another protein kinase, Fpk1, a known flippase activator. We show that Ypk1 phosphorylates and thereby down-regulates Fpk1, and further that a complex sphingolipid counteracts the down-regulation of Fpk1 by Ypk1. Our findings delineate a unique regulatory mechanism for imposing a balance between sphingolipid content and aminophospholipid asymmetry in eukaryotic plasma membranes.

[1]  E. O’Shea,et al.  Global analysis of protein expression in yeast , 2003, Nature.

[2]  R. Loewith,et al.  Tor2 Directly Phosphorylates the AGC Kinase Ypk2 To Regulate Actin Polarization , 2005, Molecular and Cellular Biology.

[3]  K. Kozminski,et al.  Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins. , 2007, Developmental cell.

[4]  G. Fink,et al.  Laboratory course manual for methods in yeast genetics , 1986 .

[5]  B. Cairns,et al.  Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7 , 1996, Molecular and cellular biology.

[6]  B. Errede,et al.  Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. , 1992, Genes & development.

[7]  Toshihide Kobayashi,et al.  Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin. , 2007, Biophysical journal.

[8]  T. Graham,et al.  Linking phospholipid flippases to vesicle-mediated protein transport. , 2009, Biochimica et biophysica acta.

[9]  T. Graham,et al.  Control of protein and sterol trafficking by antagonistic activities of a type IV P-type ATPase and oxysterol binding protein homologue. , 2009, Molecular biology of the cell.

[10]  A. Kihara,et al.  Cross talk between sphingolipids and glycerophospholipids in the establishment of plasma membrane asymmetry. , 2004, Molecular biology of the cell.

[11]  J. Thorner,et al.  Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity. , 2002, Molecular biology of the cell.

[12]  C. Paulusma,et al.  P4 ATPases - lipid flippases and their role in disease. , 2009, Biochimica et biophysica acta.

[13]  Y. Liu,et al.  The Sphingolipid Long-chain Base-Pkh1/2-Ypk1/2 Signaling Pathway Regulates Eisosome Assembly and Turnover* , 2008, Journal of Biological Chemistry.

[14]  P. Walter,et al.  Eisosomes mark static sites of endocytosis , 2006, Nature.

[15]  J. Strathern,et al.  Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual , 2005 .

[16]  Ke Liu,et al.  The Sphingoid Long Chain Base Phytosphingosine Activates AGC-type Protein Kinases in Saccharomyces cerevisiae Including Ypk1, Ypk2, and Sch9* , 2005, Journal of Biological Chemistry.

[17]  T. Hunter,et al.  The protein kinases of budding yeast: six score and more. , 1997, Trends in biochemical sciences.

[18]  Joshua D. Schnell,et al.  The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast , 2002, The Journal of cell biology.

[19]  G. Marinetti,et al.  The asymmetric arrangement of phospholipids in the human erythrocyte membrane , 1973 .

[20]  H. Riezman,et al.  Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis. , 2003, Molecular biology of the cell.

[21]  J. Thorner,et al.  Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast , 1999, Current Biology.

[22]  G. Marinetti,et al.  The asymetric arrangement of phospholipids in the human erythrocyte membrane. , 1973, Biochemical and biophysical research communications.

[23]  H. Takematsu,et al.  Sli2 (Ypk1), a Homologue of Mammalian Protein Kinase SGK, Is a Downstream Kinase in the Sphingolipid-Mediated Signaling Pathway of Yeast , 2000, Molecular and Cellular Biology.

[24]  M. Aebi,et al.  The N-glycosylation defect of cwh8Delta yeast cells causes a distinct defect in sphingolipid biosynthesis. , 2006, Glycobiology.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  T. Graham.  Flippases and vesicle-mediated protein transport. , 2004, Trends in cell biology.

[27]  Tobias Schmelzle,et al.  Yeast Protein Kinases and the RHO1 Exchange Factor TUS1 Are Novel Components of the Cell Integrity Pathway in Yeast , 2002, Molecular and Cellular Biology.

[28]  J. Thorner,et al.  Differential roles of PDK1- and PDK2-phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1 and Sch9. , 2004, Microbiology.

[29]  T. Noji,et al.  Protein kinases Fpk1p and Fpk2p are novel regulators of phospholipid asymmetry. , 2008, Molecular biology of the cell.

[30]  R. Lester,et al.  Functions and metabolism of sphingolipids in Saccharomyces cerevisiae. , 2006, Progress in lipid research.

[31]  M. Hall,et al.  Sphingoid base signaling via Pkh kinases is required for endocytosis in yeast , 2001, The EMBO journal.

[32]  L. Obeid,et al.  Yeast sphingolipids: metabolism and biology. , 2002, Biochimica et biophysica acta.

[33]  A. Kihara,et al.  Regulation of the Transport and Protein Levels of the Inositol Phosphorylceramide Mannosyltransferases Csg1 and Csh1 by the Ca2+-binding Protein Csg2* , 2007, Journal of Biological Chemistry.

[34]  T. Graham,et al.  An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal/vacuolar system. , 2002, Molecular biology of the cell.

[35]  P. Devaux,et al.  Static and dynamic lipid asymmetry in cell membranes. , 1991, Biochemistry.