The TORC2‐Dependent Signaling Network in the Yeast Saccharomyces cerevisiae

Abstract

To grow, eukaryotic cells must expand by inserting glycerolipids, sphingolipids, sterols, and proteins into their plasma membrane, and maintain the proper levels and bilayer distribution. A fungal cell must coordinate growth with enlargement of its cell wall. In Saccharomyces cerevisiae, a plasma membrane-localized protein kinase complex, Target of Rapamicin (TOR) complex-2 (TORC2) (mammalian ortholog is mTORC2), serves as a sensor and master regulator of these plasma membrane- and cell wall-associated events by directly phosphorylating and thereby stimulating the activity of two types of effector protein kinases: Ypk1 (mammalian ortholog is SGK1), along with a paralog (Ypk2); and, Pkc1 (mammalian ortholog is PKN2/PRK2). Ypk1 is a central regulator of pathways and processes required for plasma membrane lipid and protein homeostasis, and requires phosphorylation on its T-loop by eisosome-associated protein kinase Pkh1 (mammalian ortholog is PDK1) and a paralog (Pkh2). For cell survival under various stresses, Ypk1 function requires TORC2-mediated phosphorylation at multiple sites near its C terminus. Pkc1 controls diverse processes, especially cell wall synthesis and integrity. Pkc1 is also regulated by Pkh1- and TORC2-dependent phosphorylation, but, in addition, by interaction with Rho1-GTP and lipids phosphatidylserine (PtdSer) and diacylglycerol (DAG). We also describe here what is currently known about the downstream substrates modulated by Ypk1-mediated and Pkc1-mediated phosphorylation.

Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9

“…Indeed, of the 484 identified phospho-sites within a RxxS/Tmotif, 117 were significantly (P adj Ͻ 0.05) hypo-phosphorylated in at least one of the data sets. Because this is consistent with previously reported kinase consensus motifs (RRxS/T for Sch9 and PKA and RxRxxS/T for Ypk1) (51,59), we argued that the corresponding sites may constitute potential direct kinase substrates. We asked if these potential direct targets are shared between kinases tested or specific to individual kinases.…”

“…Furthermore, sites located near lysine or arginine may impede tryptic cleavage and distort the quantification of nearby residues. We therefore used the presence of arginine in the -3 position from the phosphorylated residue, which is a frequent feature of target sites of the AGC-kinases under investigation, as an additional criterion for selecting proteins with multiple regulated sites (21,59). Notable observations included the detection of two regulated RxxS-sites on the previously reported Sch9-target Stb3 in the Sch9 and PKAϩSch9 data sets (56).…”

“…Additionally, S884-phosphorylation on this protein was only affected upon Ypk1-inhibition. In the Ypk1 data set, only a few of the RxxS/T-sites on multi-site proteins were found in the strict RxRxxS/T-motif ascribed to Ypk1 (59). Instead, several of the multi-site proteins were shared with other data sets, including Smy2, Gcs1, Igo2, Ksp1, and Avt1 (supplemental Table S3).…”

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

“…For yeast cell growth, enlargement of the cell wall needs to be tightly coupled to both an increase in cell mass and expansion of the PM. TORC2-Ypk1 signaling controls the processes that maintain adequate PM levels of all of the lipid classes (sphingolipids, glycerolipids, and sterols) needed to sustain growth and viability (Roelants et al 2017a(Roelants et al , 2018. Thus, down-regulation of TORC2 activity upon CWI pathway activation provides a mechanism by which the status of the cell wall can be sensed by TORC2 and thereby the rates of the reactions necessary for PM homeostasis can be adjusted accordingly.…”

Protein kinase C: perfectly balanced

“…At the same time, PKC isozymes whose activity depended on neither Ca 2+ nor diacylglycerol, but was stimulated by phosphatidylserine (Nakanishi H and Exton 1992), were cloned (Ono et al 1988; Ono et al 1989); this class was named the atypical PKC isozymes and comprises PKCζ and PKCι/λ (human/mouse) (Nishizuka 1992). By 1992, there were nine mammalian PKC genes (Nishizuka 1992), which evolved from the single Pkc1 (corresponding not to PKC, but to the closely related PKN in mammals (Roelants et al 2017)) in Saccharomyces cerevisiae (Levin et al 1990; Watanabe et al 1994). Determination of the human kinome established that nine was, indeed, the total number of genes in the PKC family and verified that protein kinase D (PKD) (Valverde et al 1994), which had also been named PKCμ (Johannes et al 1994), was not part of the family (Manning et al 2002).…”