# The TORC2-Dependent Signaling Network in the Yeast _Saccharomyces cerevisiae_
## 4 Structure, Function, and Regulation of Ypk1
### N-Terminal Phosphorylation by Fpk1 and Fpk2  
When examined by standard SDS-PAGE, especially under conditions known to improve the resolution of phospho-isoforms (75:1 or 100:1 acrylamide: \(N\),_N_\({}^{\prime}\)-methylene-_bis_-acrylamide), Ypk1 migrates as a set of three or four distinct bands; however, none of these species could be attributed to its Pkh1- or TORC2-dependent modification, based on mutation of the corresponding sites or on inactivation of the cognate protein kinases [52]. A genome-wide screen of a protein kinase mutant collection revealed that the protein kinases responsible for the observed Ypk1 mobility shifts are Fpk1 and its paralog Fpk2/Kin82 [52]. Fpk1 phosphorylates Ser51 and Ser71 within a defined sequence context (-R-x-S>T-L/V/I/M/A-D/E-) [26; 52; 136] in the N-terminal regulatory domain of Ypk1 (Figure 1).  
Strikingly, Fpk1 is itself a substrate of Ypk1. Ypk1 also has a well-defined phosphoacceptor site specificity [-R-x-R-x-x-S>T-(Hpo)-, where (Hpo) indicates only a modest preference for a hydrophobic residue] [45; 136]. Fpk1 (893 residues) possesses three consensus Ypk1 phosphorylation motifs in its N-terminal regulatory domain (Figure 6) and the significantly shorter Fpk2 (720 residues) has one (Figure 6). Ypk1-mediated phosphorylation of Fpk1 and Fpk2 inhibits their function [52], which, in turns, blocks their ability to phosphorylate Ypk1. Thus, when cells are treated with a stimulus that activates TORC2-Ypk1 signaling, Ypk1-dependent phosphorylation of Fpk1 increases, resulting in reduced Fpk1-mediated phosphorylation of Ypk1, eliminating the slower mobility isoforms of Ypk1 observed in standard SDS-PAGE. Thus, there is an interesting reciprocal relationship between these two classes of protein kinases; and, the extent of these Ypk1 mobility shifts serves as a convenient in vivo read-out of Fpk1 (and Fpk2) activity. In this regard, Fpk1 activity in vivo depends, not only on escape from Ypk1-mediated inhibition, but also on a complex sphingolipid, namely mannosyl-inositolphosphoryl-ceramide (MIPC) [26; 52], although whether MIPC is a direct allosteric activator or acts via a more indirect mechanism has not been elucidated.  
Figure 6: Selected sites of Ypk1 phosphorylation. The phospho-acceptor sites in the indicated gene products (position of first residue in the sequence shown indicated on the left) were validated as authentic Ypk1 phosphorylation sites both in vitro and in vivo. Documentation for these assignments can be found in the following publications: Fpk1 and Fpk2 [52]; Orm1 and Orm2 [22]; Lac1 and Lag1 [23]; Gpd1 [137]; Fps1 [24]; and, Rod1 [25].  
In contrast to the clear-cut experimental evidence both in vitro and in vivo that Ypk1-mediated phosphorylation of Fpk1 inhibits its catalytic function, the effect of Fpk1-mediated phosphorylation on Ypk1 has been more difficult to discern [52]. In _S. pombe_, N-terminal phosphorylation of Gad8 (an ortholog of _S. cerevisiae_ Ypk1) on Thr6 by Pck2 (an ortholog of _S. cerevisiae_ Pkc1) reportedly prevents physical association of Gad8 with TORC2, decreases TORC2-mediated phosphorylation of its hydrophobic motif (Ser546), thus reducing TORC2-mediated activation of Gad8 [138]. Hence, it is tempting to speculate that, in the same way, Fpk1-mediated phosphorylation of Ypk1 is a negative feedback mechanism to dampen TORC2-mediated activation of Ypk1. Such a logic circuit would make physiological sense because, as discussed further below, one of the primary functions of TORC2-Ypk1 signaling is to upregulate the production of complex sphingolipids. When the supply of cellular sphingolipids is adequate, Ypk1 action is no longer needed for that purpose. Given that optimal Fpk1 function requires cellular MITC production, its phosphorylation of Ypk1 likely serves as a rheostat to adjust the level of Ypk1 activity to meet the demands of the cell for sphingolipids in a finely tuned manner.