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.

Splicing variation of BMP2K balances endocytosis, COPII trafficking and autophagy in erythroid cells

“…Based on proteomic reports (Borner et al, 2012;Brehme et al, 2009;Krieger et al, 2013) and high homology to AAK1, we initially assumed that BMP2K variants regulate endocytosis. Yeast homologues of BMP2K and AAK1 kinases, Akl1 and Prk1, suppress endocytosis (Bar-Yosef et al, 2018;Roelants et al, 2017;Takahashi et al, 2006;Zeng & Cai, 1999;Zeng et al, 2001). Consistently, we found that erythroid differentiation of K562 cells upon silencing of BMP2K expression correlated with elevated endocytic uptake.…”

The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites

“…Using budding yeast ( Saccharomyces cerevisiae ) as the experimental organism, it has been shown by us ( Roelants et al 2011 , 2017 ; Muir et al 2014 , 2015 ; Alvaro et al 2016 ) and others ( Berchtold and Walther 2009 ; Berchtold et al 2012 ; Niles et al 2012 ; Sun et al 2012 ; Fröhlich et al 2016 ) that the large multi-subunit protein kinase target of rapamycin (TOR) complex 2 (TORC2) plays an essential role in sensing and ensuring maintenance of PM homeostasis. TORC2 is one of two evolutionarily conserved TOR-containing protein complexes ( Kunz et al 1993 ; Helliwell et al 1994 ; Loewith et al 2002 ; Wedaman et al 2003 ).…”

“…TORC1 is sensitive to inhibition by rapamycin, whereas TORC2 is normally insensitive to this agent ( Jacinto et al 2004 ; Huang and Manning 2009 ; Betz and Hall 2013 ; Gaubitz et al 2015 ). In yeast, TORC2 action influences not only reactions that affect PM lipid and protein composition, but also assembly and function of the actin cytoskeleton and actin-driven endocytosis ( Bartlett and Kim 2014 ; Gaubitz et al 2016 ; Roelants et al 2017 ). The catalytic subunit of the TORC1 and TORC2 complexes is a very large TOR polypeptide; metazoans possess a single TOR-encoding gene [human mammalian TOR (mTOR), 2549 residues], whereas budding yeast ( Heitman et al 1991 ), fission yeast ( Ikai et al 2011 ), and other fungi ( Eltschinger and Loewith 2016 ) encode two TOR proteins, Tor1 and Tor2 (2470 and 2474 residues, respectively, in S. cerevisiae ).…”

“…Ypk1 reduces the rate of aminoglycerophospholipid flipping from the outer to the inner leaflet of the PM by phosphorylating and inhibiting two protein kinases, Fpk1 and Fpk2 , which stimulate the P-type ATPases (“flippases”) that catalyze this inward translocation ( Roelants et al 2010 ). Ypk1 -mediated inhibition of Fpk1 and Fpk2 also impedes endocytosis by alleviating their inhibition of the protein kinase Akl1 , which phosphorylates and blocks the function of several actin patch-associated proteins ( Roelants et al 2017 ). Ypk1 -mediated phosphorylation also blocks the ability of certain endocytic adaptors (α-arrestins) to promote internalization of integral PM proteins ( Alvaro et al 2016 ).…”

G protein α subunit suppresses sporangium formation through a serine/threonine protein kinase in Phytophthora sojae

“…In S. cerevisiae, YPK1 is directly phosphorylated by the target of rapamycin (TOR) complex-2 (TORC2) and this TOR signaling is required for many plasma membrane-and cell wall-associated events [30]. To determine whether PsGPA1 can affect the phosphorylation state of PsYPK1, we expressed PsYPK1 in wild-type, PsGPA1 overexpression or silenced strains.…”