# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 4 Structure, Function, and Regulation of Ypk1  
The _YPK1_ gene was first identified and isolated via its ability to hybridize to the complementary DNA (cDNA) for a catalytic subunit of bovine PKA [61], a founding member of the AGC-family of protein kinases [40; 41]. Likewise, a very highly related gene, initially designated _YKR2_ (now called _YPK2_), was cloned via its hybridization to a cDNA for an isoform of rabbit PKC [62], another prototypical AGC kinase. Similarly, both _YPK1_ and _YPK2_ were independently recovered using hybridization probes derived from cDNAAs encoding isozymes of rat PKCs and shown by genetic analysis to be a pair of functionally redundant loci essential for the growth of yeast cells [37]. With determination of the complete DNA sequence of the _S. cerevisiae_ genome in 1996-97 [63; 64], and now the entire genomes of many related and more distant yeasts [65; 66], it is clear that _YPK1_ and _YPK2_ are true paralogs and together represent one of the distinct classes of AGC kinases present in the fungal clade [42; 43; 44].  
Ypk1 is a 680-residue polypeptide with its catalytic domain located within its carboxy-terminal portion, preceded by a long amino-terminal segment and followed by a much shorter C-terminal extension (Figure 1). Ypk2 (677 residues) shows extensive homology to Ypk1 across its entire length and their catalytic domains share 90% sequence identity (Figure 1 legend). Aside from its homology to that in Ypk2, the N-terminal portion of Ypk1 does not exhibit detectable similarity to any characterized sequence motif or structural domain. By contrast, the C-terminal extension shares readily detectable relatedness (30% identity) to that in SGK1. To date, no regulators of Ypk1 (or Ypk2) that act via direct interaction with the N-terminal segment have been identified. However, several observations indicate that the N-terminal domain exerts some negative regulatory constraint on the C-terminal catalytic domain. First, unlike overexpression of full-length Ypk1, overexpressing an allele that lacks its entire N-terminal domain, Ypk1(\(\Delta\)2-336) (also known as Ypk1\(\Delta\)N), was toxic, whereas overexpressing a kinase-dead (K376A) derivative of Ypk1\(\Delta\)N was not [46], implying that the toxicity arises from the hyperactivity of Ypk1\(\Delta\)N. Consistent with that model, the toxicity of Ypk1\(\Delta\)N was also ameliorated in a \(pkh1\Delta\) mutant, which cripples its activation loop phosphorylation [46]. Second, a Ypk1 mutant with a single N-terminal substitution mutation (D242A) rescues the inviability of a _tor2 ts_ allele [22], in agreement with the fact that Ypk1 is a primary TORC2 target, but also suggesting that this alteration of the N-terminal domain alleviates the need for TORC2-dependent phosphorylation of Ypk1. Similarly, it was observed that a corresponding mutation in Ypk2 (D239A) [67] suppressed the growth of cells carrying the _avo3-30_ mutation, a temperature-sensitive allele that also compromises TORC2 function at the restrictive temperature [68]. These observations suggest first that, normally, the N-terminal domain interacts with and constrains the kinase fold of Ypk1 in an inactive conformation. These findings further suggest that the role of the C-terminal regulatory segment, when phosphorylated by TORC2, is to compete with and displace this N-terminal inhibitory domain (perhaps by the conformational changes induced by binding of the phosphorylated hydrophobic motif to a PIF-like pocket). If so, then the simplest model to explain the effect of the D242A mutation is that it is sufficient, by itself, to prevent the inhibitory action of the N-terminal domain.