Yeast 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Orthologs Pkh1–3 Differentially Regulate Phosphorylation of Protein Kinase A (PKA) and the Protein Kinase B (PKB)/S6K Ortholog Sch9

Abstract

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 protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated 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 disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkhdependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was 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.

Regulation of PKA activity by an autophosphorylation mechanism in Saccharomyces cerevisiae

“…In S. cerevisiae, it has been shown that Thr 241 in the Tpk1 activation loop is phosphorylated by Pkh1 immediately after the catalytic subunit is synthesized [5]. Besides Tpk1, an uncharacterized residue on Tpk2 and Thr 242 in Tpk3 are also phosphorylated in vivo by Pkh1 with different efficiency.…”

“…This phosphorylation was independent of nitrogen or glucose availability [5]. We have shown that Pkh1 inactivation reduces the interaction between Tpk1 and the regulatory subunit Bcy1 without affecting the specific kinase activity [6].…”

“…The anti-pSer 179 antibody was generated against the synthetic phosphopeptide CSLLRKpS 179 QRFPNPVAK manufactured by Pacific Immunology. Anti-pThr 241 antibody was generously provided by Professor Johan Thevelein [5]. The blots were developed with chemiluminescence luminol reagent, and immunoreactive bands were visualized by autoradiography and analysed by digital imaging using a Bio-Imaging Analyzer BAS-1800II (Fujifilm).…”

“…Recently, it has been shown that Pkh1 (homologue of mammalian PDK1) phosphorylates the activation loop of Tpk1 (Thr 241 ) in vitro, and Pkh1-Pkh3 are required for in vivo Thr 241 phosphorylation in newly synthesized Tpk1 [5]. Lack of Thr 241 phosphorylation reduces the interaction of Tpk1 catalytic subunit with the regulatory subunit Bcy1 without affecting PKA specific activity [6].…”

Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

“…These studies have shown that the Pkhs regulate a wide variety of cellular functions in S. cerevisiae, including cell wall integrity (21), sphingolipid biosynthesis (22,23), endocytosis (24), eisosome formation (25,26), flippase activity (22), and RNA metabolism (25). In S. cerevisiae, six key Pkh substrates have been identified, i.e., the AGC kinases protein kinase A (PKA), Pkc1, Ypk1/2, and Sch9 as well as a component of the eisosome, Pil1 (26)(27)(28)(29).…”