A novel HECT ubiquitin ligase regulating chemotaxis and development in Dictyostelium discoideum

Abstract

Cyclic AMP binding to G protein-coupled receptors orchestrates chemotaxis and development in Dictyostelium. By activating the RasC-TORC2-AKT/PKB module, cAMP 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. Near-saturation mutagenesis of a Pia/Rictor mutant by random gene disruption led to selection of two suppressor mutants, in which spontaneous chemotaxis and development were restored. PKB phosphorylation and chemotactic cell polarization were rescued, whereas Pia/Rictor-dependent ACA stimulation was not restored but bypassed, leading to cAMP-dependent developmental gene expression. Knocking out the gene encoding the adenylylcyclase B (ACB) in the parental strain showed ACB to be essential for this process. The gene tagged in the suppressor mutants encodes a novel HECT ubiquitin ligase, homologous to mammalian HERC1, but harbouring a pleckstrin homology domain. Expression of the isolated HECTwt, but not HECTC5185S, domain was sufficient to reconstitute the parental phenotype. The novel ubiquitin ligase appears to regulate cell sensitivity to cAMP signalling and TORC2-dependent PKB phosphorylation.