American Society for Biochemistry and Molecular Biology
Journal of Biological Chemistry vol:286 issue:25 pages:22017-22027
Pkh1, 2 and 3 are the yeast orthologs of mammalian 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast PKB and/or S6K ortholog, has been identified as one of their targets. We now show that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1 interacting fragment (PIF) pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrate that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappears during nitrogen deprivation and rapidly increases again upon nitrogen resupplementation. In addition, we show here for the first time that the PDK1 site in PKA is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh dependent in vivo and occurs during or shortly after synthesis of the PKA catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolishes binding of the regulatory subunit and cAMP dependency. As opposed to PDK1 site phosphorylation of Sch9, it is not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.