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Title: Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis
Authors: Vertommen, Didier ×
Rider, M
Ni, Y
Waelkens, Etienne
Merlevede, Wilfried
Vandenheede, Jackie
Van Lint, Johan #
Issue Date: Aug-2000
Series Title: Journal of Biological Chemistry vol:275 issue:26 pages:19567-76
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.
URI: 
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry Section (Medicine) (-)
Laboratory of Protein Phosphorylation and Proteomics
Laboratory of Phosphoproteomics (-)
× corresponding author
# (joint) last author

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