# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 6 Structure, Function and Regulation of Pkc1
### Cell Wall Integrity Pathway  
The lethal cell lysis defect of Pkc1-deficient cells provided a strong positive selection for both genomic extragenic suppressors and plasmid-borne dosage suppressors that restored growth and viability as a means to identify genes that function downstream of, but within the same pathway, as Pkc1. In this way, dominant alleles mapping to the _BCKI_ (Bypass of C Kinase) gene were identified [215]. In the immediately preceding years, the concept of multi-tiered protein kinase cascades involved in cellular responses to various stresses and extracellular stimuli had emerged from studies in both yeast and mammalian cells [252; 253; 254; 255]. The sequence similarity of Bck1 to the MAP kinase kinase kinase (MAPKKK) of the mating pheromone response pathway (Ste11) and to a MAPKKK in the HOG pathway (Ssk2), immediately suggested that one function served by Pkc1 was as the upstream activator (i.e., a MAPKKKK) of such a cascade, in the same way that Ste20 serves as the MAPKKKK in the mating pheromone response pathway [256; 257] and in the Sho1 branch of the HOG pathway [258; 259]. Indeed, a Pkc1-initiated pathway that culminates in activation of the MAPK Slt2/Mpk1 was methodically mapped out by genetic and biochemical methods, as reviewed in detail elsewhere [260; 261]. Consistent with its role as an activator of Bck1, it has been reported that Pkc1 selectively phosphorylates Bck1 in vitro at three sites (Figure 7) and, that in the absence of Bck1, Slt2/Mpk1 is not active [215; 238]. One of the reported sites in Bck1 matches well with features of the pseudosubstrate sequence in Pkc1: L at \(-5\); R at \(-3\); I/V at \(+1\); and, R or K at both \(+4\) and \(+5\) (Figure 7). Indeed, as we observed [232], and in the hands of at least two other groups, peptides in which the Ala within the pseudosubstrate sequence is mutated to either Ser or Thr serve as efficient in vitro substrates for Pkc1 purified from yeast [262; 263]. Moreover, mutational alteration of these peptides revealed that replacing the Ile at position +2 (with respect to the phosphorylated residue) with Arg enhanced phosphorylation of the peptide and its binding to Pkc1 [262; 263]. Conversely, substitution of Ala for the Arg at \(-3\) and for the basic residues at +4 and +5 eliminated phosphorylation of the peptide, and substitution of Arg \(-3\) alone had a stronger effect (100-fold poorer binding) than substitution of both basic residues at +4 and +5 alone (10-fold reduction in binding) [262]. On this basis, it appears that the minimal phospho-acceptor site preference for Pkc1 is R (or K) at \(-3\) and R (or K) at +2 [263]. The other two reported sites in Bck1 fit this consensus (Figure 7). Unlike _pkc1\(\Delta\)_ mutants, _slt2\(\Delta\)/mpk1\(\Delta\)_ cells are viable under normal growth conditions [264], indicating that Pkc1 action is required for other cellular processes in addition to its function in stimulating the cell wall integrity (or, perhaps, better called the \"cell wall damage repair\") pathway [228]. As one means to detect additional potential Pkc1 substrates, MS analysis was conducted [265] comparing normal cells to those overexpressing a constitutively-active Pkc1 allele, Pkc1(R398A R405A K406A), dubbed Pkc1*, that is hyper-active because critical residues necessary for the inhibitory function of its pseudosubstrate sequence have been mutated [263]. Numerous phosphopeptides representing both validated and potentially new Slt2/Mpk1 targets were identified by this procedure; but, surprisingly, no phosphopeptides corresponding to Bck1 or to any other of the putative Pkc1 substrates reported by others (Figure 7) were recovered by this in vivo approach [265].