# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 6 Structure, Function and Regulation of Pkc1
### Modulation by PtdSer and DAG  
Shortly after the _PKC1_ gene was cloned, a C-terminally epitope-tagged version was expressed in yeast and recovered from cell extracts by immuno-precipitation under non-denaturing conditions, presumably with Rho1 still bound, although that was not examined at the time (232). Using these preparations for enzyme assays, calf thymus histones and myelin basic protein as surrogate substrates (because no physiologically relevant substrate for Pkc1 was then known), and Mgand [\(\gamma\)-\({}^{32}\)P]ATP, it was found that addition of Ca\({}^{2+}\), PtdSer, and DAG synergistically stimulated Pkc1 autophosphorylation as well as incorporation of phosphate into these substrates [232]. Moreover, addition of a synthetic peptide that mimics the pseudosubstrate sequence in Pkc1 (except that the Ala was changed to a Ser) (Figure 7), namely GGLHRHG(A \(\rightarrow\) S)IINRK, markedly reduced both autophosphorylation and substrate phosphorylation [232]. Quite recently, this issue was revisited for protein substrates thought to be bona fide in vivo targets of Pkc1. For peptides containing sites from Pah1 (PtdOH phosphatase), Nem1 (catalytic subunit of a protein phosphatase that dephosphorylates Pah1), and Spo7 (regulatory subunit of the protein phosphatase that dephosphorylates Pah1), the efficiency of their phosphorylation was not enhanced by the presence of lipids [233]. However, when the same full-length proteins (Pah1, Nem1, and Spo7) were used as substrates, PtdSer and DAG were required for their phosphorylation, with PtdSer having a greater effect than DAG [233]. Moreover, in an in vitro liposome binding assay, the presence of PtdSer enhanced Pkc1 retention in a dose-dependent manner and, in _cho1\(\Delta\)/pss1\(\Delta\)_ cells (which lack PtdSer synthase), the degradation of Pah1, which is thought to be a Pkc1-dependent process, was attenuated, consistent with a role for PtdSer in regulating Pkc1 function in vivo [233]. In this same regard, Pkc1 contains an apparent C2 domain (Figure 2), which is net acidic (20 D and E, 16 R and K) and with three of the five residues equivalent to those that chelate Ca\({}^{2+}\) in synaptotagmin conserved (E234, D277, and D280) and the other two positions neutral (M228 and V279) [234]. C2 domains with these features have been amply demonstrated to mediate binding to anionic lipids on the cytosolic surface of the PM, especially PtdSer and phosphoinosititides (PIPs) in a Ca\({}^{2+}\)-dependent manner [235; 236]. Thus, as in other C2 domain-containing proteins, it is interaction with this region of Pkc1 that presumably mediates the observed stimulatory effect of PtdSer. Furthermore, unlike the inviability of _pkc1\(\Delta\)_ cells, the temperature-sensitive lethality of each of three different _pkc1\({}^{ts}\)_ alleles [Pkc1(N834I), Pkc1(L887S), and Pkc1(P1023L)] could be suppressed on medium containing 25 mM CaCl\({}_{2}\)[213], at least consistent with the stimulatory effect of the presence of Ca\({}^{2+}\) on Pkc1 activity in vitro that we observed [232]. Pkc1 (WS) 389 RKQLMGGLH HGALT RREEIFEQRH  
Bck1 1122 TIHAEASLAKNSVKKKKKKONTRWVG  
Bck1 1107 PLNQAKTPKKKRTRILHAEASLAR  
Bck1 927 FYPSPSPLHKRSNSKKWSSTSAADI  
Ncd1 508 GNNSNSNNVKKASKKKNSQSSLLSD  
Neml 189 RDNVKGSILROSKKFRYSKSSE  
Pahl 757 MDDEDSNYNKTSKKKASAATSID  
Ura7 24 GMLMKTLGKTSKKIDYMNIDAG  
Apm4 164 SSAGELHFFKLTRRSSFLGQQSDS  
Syp1 335 KKSSHTLRSKGSIFKKTRNKQ  
Hht1/2 34 GGVKKPRHKQFGTKREIRRFQKS  
Nddl 515 TKRRASKLRRSK  
often commercially-available preparations of mammalian PKCs were used to define \"Pkc1\" sites in other yeast proteins, instead of bona fide purified and Rho1-GTP-activated Pkc1. Moreover, for many of these putative sites, the phosphorylated residue was not unequivocally identified nor was its modification demonstrated to be Pkc1-mediated in vivo. So, many of these assignments need to be verified. Furthermore, spatial restriction of Pkc1 and/or of its required cofactors will dictate in which subcellular compartment Pkc1 can act and therefore what substrates it can phosphorylate.