# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 4 Structure, Function, and Regulation of Ypk1
### C-Terminal Phosphorylation by TORC2  
Pkh1- and Pkh2-mediated phosphorylation of the activation loop in Ypk1 and Ypk2 is sufficient to confer basal activity. However, for cell survival in response to certain stresses (e.g., sphingolipid depletion, heat shock, hypotonic conditions, high exogenous acetic acid), Ypk1 activity must be upregulated further by phosphorylation at Thr662 within its conserved hydrophobic motif sequence near its C-terminus [22; 28; 29; 30; 47; 67; 111; 112] (Figures 1 and 5). As first revealed by analysis of Ypk2 [67], phosphorylation at the hydrophobic motif is mediated by TORC2, which also phosphorylates another C-terminal site (Ser644 in Ypk1) within another conserved sequence, dubbed the turn motif [47; 67] (Figure 5). Crystal structures of mammalian AGC kinases have revealed that, when phosphorylated, the hydrophobic motif docks in the PIF pocket in the N-lobe of the kinase domain [55; 113] and, likewise, when phosphorylated, the turn motif interacts with another positively-charged pocket in the N-lobe [114; 115]. In both cases, these interactions induce restructuring of the kinase domain, stabilizing it in the conformation found in the fully active state. Figure 4: Sites phosphorylated by Pkh1 and Pkh2. The activation loop sequence in the indicated _S. cerevisiae_ protein kinases (phosphorylated by Pkh1 and Pkh2) and their corresponding human counterparts (phosphorylated by PDK1), a putative Pkh1 site in the fungal-specific protein Pill, and a purported Pkh1 site in the ESCRT-0 subunit Vps27 were aligned by anchoring on the residue phosphorylated (bold red). Identities (white letters on black boxes) and standard conservative substitutions (bold letters on grey boxes), and a one-residue gap (hyphen) to maximize one alignment, are indicated. Period (.) indicates the end of the open-reading-frame. Sequence sources were the Saccharomyces Genome Database (SGD; [[http://www.yeastgenome.org](http://www.yeastgenome.org)]([http://www.yeastgenome.org](http://www.yeastgenome.org)); Stanford University, CA, USA) and the ProteomeTM Database of geneXplain GmbH ([[https://portal.genexplain.com/cgi-bin/portal/login.cgi](https://portal.genexplain.com/cgi-bin/portal/login.cgi)]([https://portal.genexplain.com/cgi-bin/portal/login.cgi](https://portal.genexplain.com/cgi-bin/portal/login.cgi)); Wolfenbuttel, Germany). Recently, we have shown that several additional sites in Ypk1, aside from the turn and hydrophobic motif, are also phosphorylated by TORC2 (Figure 5) and that these sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662 [39]. Moreover, phosphorylation at these sites is a prerequisite for TORC2 phosphorylation of Thr622 in the hydrophobic motif [39]. Quite similarly, the mammalian AGC kinase AKT1 is phosphorylated in an mTOR2-dependent manner at other C-terminal sites that are necessary for efficient phosphorylation of its hydrophobic motif [116]. Furthermore, phosphorylation at the turn motif has been shown to be especially important for proper carboxyl-terminal folding and stability of mammalian AKT and PKC [117; 118]. On this basis, it seems likely that phosphorylation of the newly identified TORC2 sites in Ypk1 is also a prelude to phosphorylation of Ser644 in its turn motif because absence of phosphorylation at the new sites markedly reduces Ypk1 stability. These observations make it clear that TORC2 plays a key role in stimulating Ypk1 activity, up to the level needed by the cell to cope with the stresses of sphingolipid depletion, heat shock, hypotonicity, and other stressful conditions known to perturb PM structure and/or function. There has been some degree of controversy about how TORC2 recognizes Ypk1 (and Ypk2) as substrates. Two groups have reported [28; 29] that Ypk1 is delivered to the catalytic core  
Figure 5: Conservation of sites phosphorylated by TORC2 in Ypk1.