title: TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae.
abstract: Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Françoise M
authors.initials: FM
authors.lastname: Roelants
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Kristin L
authors.initials: KL
authors.lastname: Leskoske
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Ross T A
authors.initials: RTA
authors.lastname: Pedersen
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Muir
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Jeffrey M-H
authors.initials: JM
authors.lastname: Liu
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Finnigan
authors.affiliation: Divisions of Biochemistry, Biophysics & Structural Biology and Cell & Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA jthorner@berkeley.edu.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D000595:Amino Acid Sequence; D015536:Down-Regulation; D004705:Endocytosis; D000076225:Mechanistic Target of Rapamycin Complex 2; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D055550:Protein Stability; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013107:Sphingolipids / Q000378:metabolism; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; D010768:Phosphoserine; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases
keywords: homeostasis; mutants; phosphorylation; plasma membrane; stress response; yeast