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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. [ABSTRACT FROM AUTHOR]

Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is a multi-subunit plasma membrane-associated protein kinase and vital growth regulator. Its essential functions are exerted via phosphorylation and stimulation of downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 phosphorylates multiple substrates to regulate plasma membrane lipid and protein composition. Ypk1 function requires phosphorylation of Thr504 in its activation loop by eisosome-associated Pkh1 (and its paralog Pkh2). For cell survival under certain stresses, however, Ypk1 activity requires further stimulation by TORC2-mediated phosphorylation at C-terminal sites, dubbed the "turn" (Ser644) and "hydrophobic" (Thr662) motifs. Here we show that four additional C-terminal sites are phosphorylated in a TORC2- dependent manner, collectively defining a minimal consensus. We found that the newly identified sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A), which has been demonstrated to bypass the need for TORC2-mediated phosphorylation, restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-dependent activation of Ypk1. [ABSTRACT FROM AUTHOR]