# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 4 Structure, Function, and Regulation of Ypk1
### Activation Loop Phosphorylation by Pkh1 and Pkh2
In addition to their interaction with Pil1, it has been reported that both Pkh1 and Pkh2 are able to bind directly in vitro to two distinctly different types of negatively-charged lipids, PtdIns4,5P2 and the phosphorylated form of the sphingoid long-chain base dihydrosphingosine-1-phosphate (DHS-P) [70] and, further, that these interactions are synergistic [93]. If the cooperative effect of these two lipids is crucial for either the localization or function of Pkh1 and/or Pkh2, and thus for their ability to activate their downstream targets, it may explain, at least in part, why production of PtdIns4,5P2 is essential for yeast cell growth [94] as well as reports that DHS-P production is needed for maximal growth rate and for survival in response to heat stress [95; 96]. In this same regard, it was reported some time ago that Pkh1 and Pkh2 can be stimulated by the long-chain base PHS in vitro [97], and another group claimed to see the same effect [98; 99], raising speculation that PHS (or a derived product) serves as a \"second messenger\" emanating from the sphingolipid pathway that is critical for Pkh1- and Pkh2-mediated Ypk1 activation [100; 101]. However, using a phosphosite-specific antibody that we demonstrated only recognizes Ypk1 phosphorylated at its Pkh1- and Pkh2-dependent site (T504) [45; 52], we found that neither blocking production of endogenous PHS (by myriocin treatment) nor elevating intracellular PHS (by supplying copious exogenous PHS) had any effect on the level of T504 phosphorylation in vivo [52]. Thus, there is  
Figure 3: Comparison of Pkh1 and Pkh2. Catalytic domain (yellow), N- and C-terminal extensions (white) and position of a putative lipid-binding domain (blue) characterized in Pkh2 only [70; 71; 72] are only indicated. no credible, physiologically relevant evidence that, in the cell, activation loop phosphorylation of Ypk1 by Pkh1 and Pkh2 is controlled by PHS or any derived product. Thus, the conclusion that Pkh1 and Pkh2 are \"sphingolipid-regulated\" protein kinases, a concept which seems to continue to be propagated in the literature [70; 102], is not correct. In any event, using both a lipid overlay assay and a flotation assay to assess binding to liposomes, the putative PtdIns4,5P2-binding element in Pkh2 has been localized to a \(\sim\)300-residue \"PH domain-like\" segment of its C-terminal extension [71; 72]. Curiously, however, despite the reported similarity in lipid-binding properties of Pkh1 and Pkh2 [70], there is very little sequence relatedness between the purported lipid-binding element in Pkh2 and the corresponding region in Pkh1 (Figure 3). Two transmembrane proteins that interact with each other, Sng1 and Nce102, the latter of which localizes to eisosomes, reportedly also contribute to optimal Pkh1 and Pkh2 signaling [103; 104], but the mechanism by which they do so is unclear. Given the tight association of Pkh1 and Pkh2 with eisosomes, all of the target AGC protein kinases that they phosphorylate--including Pkcl [47; 69]; Ypk1 and Ypk2 [46; 47; 98]; Sch9 [47; 56; 98]; and Tpk1, Tpk2, and Tpk3 [105] (Figure 4)--must each have some mechanism to bring them to the PM for their encounter with these upstream activators. Before it was appreciated that Pil1 and Lsp1 were the primary components of eisosomes, it was claimed that they served as negative regulators of Pkh1 and Pkh2 activity [85]; however, these conclusions were based on Ypk1 mobility shift assays that were presumed to be due to its Pkh1-mediated phosphorylation, but were later shown to be due to mobility shifts arising from phosphorylation of Ypk1 at sites phosphorylated by the protein kinases Ppk1 and Fpk2 [52] (Figure 1), which are discussed further below. Pil1 itself is, however, an apparent substrate of Pkh1, and its purported Pkh1-dependent modification has some influence on eisosome assembly and organization [85; 99; 106].