# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 3 TORC2 Effects
Abbreviations: Hr1 (pink), Homology region 1, a compact helical bundle, also referred to as an ACC (anti-parallel coiled-coil) finger domain, which binds small GTPases of the Rho family; C2 (yellow), Conserved region 2, an 8-stranded \(\beta\)-sandwich rich in acidic residues, also referred to as a CalB (Ca\({}^{2+}\)-binding) domain, which binds to phospholipids, especially PtdSer, in a Ca\({}^{2+}\)-dependent manner; YS (red), pseudosubstrate sequence, a short sequence that lacks a phosphoacceptor residue, but otherwise possesses the features of the optimal Pkc1 consensus phosphoacceptor site, which binds to and occludes the catalytic pocket in the kinase domain, thereby keeping the enzyme inactive; C1 (green), Conserved region 1, a Cys-rich Zn\({}^{2+}\)-binding fold with the capacity to bind 1,2-DAG or their pharmacological mimic, phorbol esters; CC (brown), a sequence with a strong propensity to form a coiled-coil, and LX (gray), a low-complexity sequence, as predicted by UnitProt [50] and/or SMART [51] databases; AL, activation loop Thr (T983), phosphorylated by Pkh1 (and Pkh2); TM, turn motif Thr (T1125), and, HM, hydrophobic motif Ser (S1143), both phosphorylated by TORC2. phosphorylate the activation loop of the bound AGC kinase (a proximity effect) more efficiently. On the contrary, analysis of crystal structures for the prominent AGC kinase family member AKT1/PKB suggested a different mechanistic explanation for why hydrophobic motif phosphorylation precedes activation loop phosphorylation. These studies showed that AKT1 also possesses a PIF pocket and, when its hydrophobic motif (FPQFSYSAS) is phosphorylated, it can dock in the PIF pocket in an intramolecular fashion [55]. This interaction causes a disorder-to-order transition that structures important elements of the kinase domain, including stabilizing the \(\alpha\)C helix and reconfiguring the activation loop [55]. This dramatic conformational change of the activation loop facilitates its recognition and phosphorylation by PDK1 (an accessibility effect). In any event, for Ypk1, phosphorylation of its hydrophobic motif (FGGWT662 YVGN) cannot be an obligatory prelude to Pkh1-dependent phosphorylation of its activation loop based on the following findings. A Ypk1(T504A) mutant, which cannot be and is not phosphorylated on its activation loop [52], is non-functional in vivo because it was unable to support growth, whereas a Ypk1(T662A) mutant supported cell viability as efficiently as wild-type (WT) Ypk1 under standard growth conditions, i.e., on rich medium at 30 \({}^{\circ}\)C [47]. Moreover, purified Ypk1(T504A) was catalytically inactive in vitro [45], whereas Ypk1(T662A) was only slightly less active than normal Ypk1 [47]. Nonetheless, when isolated from yeast cells, a phosphomimetic allele, Ypk1(T662D), reproducibly exhibited a two-fold higher specific activity, as compared to WT Ypk1 or Ypk1(T662A) [47]. Hence, it is possible that Pkh1 possesses the equivalent of a PIF pocket and the negative charge in the hydrophobic motif in Ypk1(T662D) mimics phosphate, permitting higher affinity recruitment of Pkh1 and more efficient activation loop phosphorylation. In fact, all of the residues in mammalian PDK1 that constitute its PIF pocket (including the phosphate-binding residues) are conserved in yeast Pkh1 and Pkh2 [47; 56]. Alternatively, however, like AKT1, Ypk1 may itself contain a PIF pocket and, when it is occupied by the phosphorylated hydrophobic motif, or the phospho-mimetic version in Ypk1(T662D), it reinforces a conformation that is intrinsically more catalytically active. We favor this latter model, as discussed further below. The _PKC1_ gene also has an apparent human ortholog. _PKC1_ was so designated, at the time, because of its resemblance to members of another class of AGC-family protein kinase, the \"conventional\" mammalian PKC family [38]. However, this classification is a misnomer.