# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 5 Substrates of Ypk1
### Other Potential Targets  
The MADS-box transcription factor Smp1, paralog of Rlm1 [183; 184], was identified initially as a potential Ypk1 target because it was isolated as a dosage suppressor of the temperature-sensitive phenotype of \(ypk1^{ts}\)\(ypk2\Delta\) cells [46]. It was identified independently as a likely Ypk1 substrate in our global screen [23] and confirmed to be an authentic substrate of Ypk1 in vivo [185]. Smp1 has been implicated both in response to hyperosmotic stress [184; 186] and in sensitivity to the toxic effects of high exogenous Fe\({}^{3+}\)[187] and, both Smp1 and Ypk1 are required for iron toxicity [187]. Preliminary microarray analysis [188] supports the conclusion that TORC2-Ypk1 signaling may be mechanistically coupled to iron metabolism by modulation of genes under Smp1 transcriptional control. Ypk1 may also contribute to translational control, given that phosphorylation of Ser232 in ribosomal protein S6 (Rps6) has been attributed, in part, to Ypk1 action [189]. In this regard, it has also been reported [190] that loss of Ypk1 function impedes translation, apparently due to depletion of eIF4G, an initiation factor essential for cap-dependent mRNA translation [191]. There is also circumstantial evidence that the actions of Ypk1 and/or Ypk2 have roles in various other aspects of yeast cell physiology, including dampening the damaging effects of reactive oxygen species (ROS) from both vacuolar and mitochondrial sources [192; 193], as well as promoting autophagy in a Ca\({}^{2+}\)-dependent manner when cells are starved specifically for amino acids [194; 195]. The mechanism by which Ypk1 regulates ROS is unclear, but somehow involves Ypk1 control of flippases and sphingolipids [192; 193]. In addition, Ypk1 reportedly modulates the activity specifically of the Tpk3 isoform of PKA, by somehow upregulating expression of \(PDE2\), a gene encoding the high-affinity 3',5'-cyclic-AMP phosphodiesterase [193]. However, in none of these studies has the actual substrate phosphorylated by Ypk1 and/or Ypk2 that is responsible for the reported phenotypic observations been pinpointed. Also, in a study of ligand-induced endocytosis of Ste2 [196], it was reported that \"purified\" Ypk2 (but not putative kinase-dead mutants) was able to phosphorylate in vitro a site (Ser357) in a fragment of the ATPase domain of the paralogous type I myosins Myo3 and Myo5, which serve as the motor proteins for actin-driven endocytosis, and that this modification promotes Ste2 internalization [197]. This conclusion seems suspect, however, given that it was not shown that modification of this site in vivo is Ypk2-dependent under any condition and given the more recent evidence, summarized in a preceding section, that Ypk1 and Ypk2 negatively regulate endocytosis. Moreover, this same site was well-documented long ago to be the target for the yeast Cdc42-dependent protein kinases Cla4 and Ste20 (homologs of mammalian p21-activated protein kinases / PAKs) [198]. Indeed, Ser357 in Myo3 and Myo5 lies in a sequence context that was shown by others to be the optimal consensus phospho-acceptor motif for Cla4 and Ste20 [136; 199; 200], and certainly not for Ypk1 and Ypk2 [23; 136]. Another means to glean what aspects of cell function are under TORC2-Ypk1 control was a so-called chemical genetic approach [201]. Specifically, Tor2 was mutagenized and a derivative was isolated that is preferentially susceptible to inhibition by a chemical TOR inhibitor, the imidazoquinoline derivative NVP-BEZ235 [202]. This tool--the ability to selectively inhibit TORC2 action--was combined with a collection of deletion mutants to identify what processes, when eliminated, are especially deleterious to cell growth and survival when TORC2 action (and presumably Ypk1 activity) is limiting. This analysis suggested some connection between TORC2 action and the pentose-phosphate pathway [202], in keeping with the growth-promoting role of TORC2 and the demand for NADPH in many cellular anabolic reactions.