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.
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December 2012
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Research Article|
November 21 2012
The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae
Steven Haesendonckx;
Steven Haesendonckx
1
*Department of Molecular Biology, University of Geneva CH-1211, Switzerland
†Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31. B-3001 Leuven-Heverlee, Flanders, Belgium
‡Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders, Belgium
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Vanesa Tudisca;
Vanesa Tudisca
1
§Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires 1428, Argentina
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Karin Voordeckers;
Karin Voordeckers
†Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31. B-3001 Leuven-Heverlee, Flanders, Belgium
‡Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders, Belgium
∥VIB Laboratory for Systems Biology, Gaston Geenslaan 1, B-3001 Leuven, Belgium
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Silvia Moreno;
Silvia Moreno
§Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires 1428, Argentina
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Johan M. Thevelein;
Johan M. Thevelein
†Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31. B-3001 Leuven-Heverlee, Flanders, Belgium
‡Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders, Belgium
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Paula Portela
Paula Portela
2
§Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires 1428, Argentina
2To whom correspondence should be addressed (email pportela@qb.fcen.uba.ar).
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Publisher: Portland Press Ltd
Received:
July 02 2012
Revision Received:
September 06 2012
Accepted:
September 07 2012
Accepted Manuscript online:
September 07 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 448 (3): 307–320.
Article history
Received:
July 02 2012
Revision Received:
September 06 2012
Accepted:
September 07 2012
Accepted Manuscript online:
September 07 2012
Citation
Steven Haesendonckx, Vanesa Tudisca, Karin Voordeckers, Silvia Moreno, Johan M. Thevelein, Paula Portela; The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae. Biochem J 15 December 2012; 448 (3): 307–320. doi: https://doi.org/10.1042/BJ20121061
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