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Scientific article
Open access
English

Target of rapamycin complex 2–dependent phosphorylation of the coat protein Pan1 by Akl1 controls endocytosis dynamics in Saccharomyces cerevisiae

Published inThe Journal of biological chemistry, vol. 293, no. 31, p. 12043-12053
First online date2018-08
Abstract

Target of rapamycin complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In the yeast Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. In this role, TORC2 regulates diverse processes, including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton, and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here, we report that Fpk1 mediates TORC2 signaling to control actin polarization, but not endocytosis, via aminophospholipid flippases. To search for specific targets of these flippase kinases, we exploited the fact that Fpk1 prefers to phosphorylate Ser residues within the sequence RXS(L/Y)(D/E), which is present ∼90 times in the yeast proteome. We observed that 25 of these sequences are phosphorylated by Fpk1 in vitro. We focused on one sequence hit, the Ark/Prk-family kinase Akl1, as this kinase previously has been implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to preferentially inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which, in turn, reduces phosphorylation of Pan1 and of other endocytic coat proteins and ultimately contributes to a slowing of endocytosis kinetics. These results indicate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled through separate pathways that bifurcate at the level of the flippase kinases.

eng
Keywords
  • Ark1/Prk1 family
  • Fpk1
  • Pan1
  • Actin
  • Aminophospholipid flippase
  • Endocytosis
  • In vitro kinase assay
  • Membrane function
  • Signal transduction
  • Target of rapamycin (TOR)
  • Actin Cytoskeleton / drug effects
  • Actin Cytoskeleton / metabolism
  • Actin Cytoskeleton / ultrastructure
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Endocytosis / drug effects
  • Endocytosis / genetics
  • Gene Expression Regulation, Fungal
  • Glycerol / metabolism
  • Glycogen Synthase Kinase 3 / genetics
  • Glycogen Synthase Kinase 3 / metabolism
  • Mechanistic Target of Rapamycin Complex 2 / antagonists & inhibitors
  • Mechanistic Target of Rapamycin Complex 2 / genetics
  • Mechanistic Target of Rapamycin Complex 2 / metabolism
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / ultrastructure
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Serine / metabolism
  • Signal Transduction
  • Sphingolipids / biosynthesis
Citation (ISO format)
BOURGOINT, Clelia et al. Target of rapamycin complex 2–dependent phosphorylation of the coat protein Pan1 by Akl1 controls endocytosis dynamics in <i>Saccharomyces cerevisiae</i>. In: The Journal of biological chemistry, 2018, vol. 293, n° 31, p. 12043–12053. doi: 10.1074/jbc.RA117.001615
Main files (1)
Article (Published version)
Secondary files (1)
Identifiers
ISSN of the journal0021-9258
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