Author:

Keywords:

Alanine, Alkaline Phosphatase, Binding Sites, Cell Line, Down-Regulation, Electrophoresis, Polyacrylamide Gel, Enzyme Inhibitors, Glutamic Acid, Humans, Indoles, Kinetics, Maleimides, Models, Biological, Mutagenesis, Site-Directed, Phosphorylation, Precipitin Tests, Protein Kinase C, Recombinant Proteins, Research Support, Non-U.S. Gov't, Signal Transduction, Spectrum Analysis, Mass, Time Factors, Transfection, Trypsin, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, SIGNAL-TRANSDUCTION PATHWAY, PLECKSTRIN HOMOLOGY DOMAIN, C-MU, PHORBOL ESTERS, D ACTIVATION, PROTEOLYTIC ACTIVATION, CATALYTIC DOMAIN, STRUCTURAL BASIS, PKD ACTIVATION, INTACT-CELLS, Mass Spectrometry, 03 Chemical Sciences, 06 Biological Sciences, 11 Medical and Health Sciences, 31 Biological sciences, 32 Biomedical and clinical sciences, 34 Chemical sciences

Abstract:

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.