UNTANGLING THE COMPLEXITY OF TORC2 REGULATION BY USING PIA/RICTOR MUTANTS

In Dictyostelium cells the activation of the RasC-TORC2-AKT/PKB module, upon cAMP binding to its cognate receptor, regulates cell polarization during chemotaxis. TORC2 also mediates GPCR-dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 PIA/RICTOR subunit are impaired in chemotaxis and development. The HSB1 is a temperature-sensitive mutant harbouring a point mutation (G917D) within the PiaA /Rictor gene (Pergolizzi et al. 2002). Near-saturation mutagenesis of HSB1 led to isolation of a suppressor mutant, named HSB1HECTPH1- , in which spontaneous chemotaxis and development were restored. TORC2-dependent PKBs phosphorylation/activity and chemotactic cell polarization were rescued on the contrary the PIA-dependent ACA stimulation was not reestablished but bypassed, leading to spontaneous chemotaxis and cAMP-dependent developmental gene expression (Pergolizzi et al., 2017). Unlike most of yeast TORC2 suppressor mutants, in which the rescued phenotype is ascribable to a constitutive activation of TORC2 effector/s, i.e. AGC kinase family members, the analysis of the HSB1HECTPH1- reveals that the gene responsible for the phenotype reversion encodes for a HECT ubiquitin ligase, named HECTPH1, homologous to mammalian HERC1, but containing a PH domain, and being the first ubiquitin ligase suppressing TORC2 deficiency. The mutant analysis indicates that the HECTPH1regulates/controls cell sensitivity to cAMP signalling and TORC2-dependent PKB phosphorylation. How, at what level/s and to what extent the inactivation of the ubiquitin ligase contributes to the phenotype reversion remains to be ascertained. HSB1HECTPH1- displays rescued PKB activity, but to what degree the Pia G917D mutation affects TORC2 integrity/assembly is unknown. A single point mutation of the Rictor gene - G1120E and G934E - in C. elegans and mammals, respectively, results in Rictor deficiency. Since the 917 Gly residue is conserved throughout evolution, corresponding to the human RICTOR codon 884, we undertook a functional study aimed to assess whether the Pia G917D mutation affects TORC2 integrity in Dictyostelium and mammalian cells. We show that the point mutation G917D impairs TORC2 assembly/integrity in Dictyostelium, whereas the corresponding mutation in mammalian cells has no such effect.