title: The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae.
abstract: PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates PKA (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the PKA catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an HM-PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized, Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.
authors.affiliation: Department of Molecular Biology, University of Geneva CH-1211, Switzerland.
authors.forename: Steven
authors.initials: S
authors.lastname: Haesendonckx
authors.forename: Vanesa
authors.initials: V
authors.lastname: Tudisca
authors.forename: Karin
authors.initials: K
authors.lastname: Voordeckers
authors.forename: Silvia
authors.initials: S
authors.lastname: Moreno
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein
authors.forename: Paula
authors.initials: P
authors.lastname: Portela
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000502:physiology*; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000378:metabolism*; D054743:Cyclic AMP-Dependent Protein Kinase Type I / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology*
chemical_list: D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D064413:3-Phosphoinositide-Dependent Protein Kinases; C118385:PKH1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D054743:Cyclic AMP-Dependent Protein Kinase Type I; D017868:Cyclic AMP-Dependent Protein Kinases; C457179:TPK2 protein, S cerevisiae; C509333:Tpk3 protein, S cerevisiae