# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 4 Structure, Function, and Regulation of Ypk1
### Activation Loop Phosphorylation by Pkh1 and Pkh2  
As observed for other AGC family protein kinases [40; 41], activation of Ypk1 is regulated by phosphorylation on residues situated within three conserved sequences. First, for any activity at all, Ypk1 must be phosphorylated on its activation loop (T-loop) at Thr504 within a conserved \(\textbf{T}^{504}\)FCGTPEY motif, a modification installed, as mentioned above, by Pkh1 and Pkh2 [46; 47]. Pkh1 (766 residues) and the significantly larger Pkh2 (1081 residues) are only closely related within their catalytic domains (72% identity) (Figure 3). So-called Pkh3, which was isolated as a high-copy-number (dosage) suppressor of the temperature-sensitive growth defect of a _pkh1(D398G)ts_pkh2\(\Delta\) double mutant [69], is intermediate in size (898 residues), has a different organization (its catalytic domain is situated at its immediate N-terminal end), and bears substantially less relatedness (46% identity) to either Pkh1 or Pkh2 and only in its kinase domain. Moreover, unlike the inviability of _pkh1\(\Delta\) pkh2\(\Delta\)_ cells, a _pkh3\(\Delta\)_ single mutant and _pkh1\(\Delta\) pkh3\(\Delta\)_ and _pkh2\(\Delta\) pkh3\(\Delta\)_ double mutants grew comparably to WT cells under all conditions tested [69]. Furthermore, high-level expression of _PKH3_ was unable to rescue the inviability of _pkh1\(\Delta\) pkh2\(\Delta\)_ cells [69], indicating, first, that the _pkh1(D398G)ts_ allele must retain some physiological function in the cell even at a restrictive temperature and, second, that Pkh3 cannot truly compensate for the lack of Pkh1 and Pkh2. Pkh1 and Pkh2 are tethered to the PM via their tight association with Pil1, a phosphatidylinositol-4,5-_bi_sphosphate (PtdIns4,5P2)-binding protein containing an N-terminal region of homology to the human proteins BIN1 and Amphiphysin and the yeast protein Rvs167 (BAR domain) that is most similar to that in human proteins FER and CIP4 (F-BAR domain) [73], and its paralog Lsp1 [74], which are primary subunits of peripherally PM-bound protein complexes, dubbed eisosomes [75; 76], that bear certain similarities to mammalian caveoli [77]. In fact, the interaction between Pkh1 and Pil1 is so robust that it was detected in two independent genome-wide two-hybrid screens [78; 79], as well as in two independent genome-wide analyses of isolated tandem affinity purification (TAP)-tagged protein complexes by mass spectrometry (MS) [80; 81]; and, in our hands, Pkh1 and Pil1 can be readily and reciprocally co-immunoprecipitated [82]. Initially, eisosomes were thought to be special sites of endocytosis [75], distinct from the clathrin- and actin-dependent route [83], but further analysis indicated that eisosomes are not sites for endocytic entry [84]. Given that _pil1_ mutants lack normal eisosomes and that even _pil1_ _lsp1_ double mutants are viable [75; 85], the exact role of these structures in cell physiology has remained elusive. Because Pil1 and Lsp1 binding causes the PM to invaginate, it has been suggested that these structures serve as reservoirs of \"extra\" membrane material on which the cell can draw when it needs to expand rapidly, e.g., under hypotonic conditions [86]. It has also been suggested that, aside from PtdIns4,5P2 binding by Pil1 and Lsp1, eisosomes have another function in maintaining the level of PtdIns4,5P2 in the inner leaflet of the PM because it has been observed that Inp51, one of three synaptojanin-like PtdIns4,5P2 5-phosphatases in _S. cerevisiae_[87; 88], specifically associates with Pil1, thereby recruiting this enzyme to the PM [89]. On the other hand, it has been reported previously that, instead, Inp51 is recruited to the PM via specific interaction with Tax4 (and its paralog Irs4), which are orthologs of mammalian EPS15 [90]. Similarly, in _S. pombe_, there is evidence that eisosomes are involved in regulating PM PtdIns4,5P2 levels [91] and localization [92].