Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis

J Biol Chem. 2000 Jun 30;275(26):19567-76. doi: 10.1074/jbc.M001357200.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry
  • Alkaline Phosphatase / pharmacology
  • Binding Sites
  • Cell Line
  • Down-Regulation
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology
  • Glutamic Acid / chemistry
  • Humans
  • Indoles / pharmacology
  • Kinetics
  • Maleimides / pharmacology
  • Mass Spectrometry
  • Models, Biological
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Precipitin Tests
  • Protein Kinase C / metabolism*
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Time Factors
  • Transfection
  • Trypsin / metabolism


  • Enzyme Inhibitors
  • Indoles
  • Maleimides
  • Recombinant Proteins
  • Glutamic Acid
  • protein kinase D
  • Protein Kinase C
  • Alkaline Phosphatase
  • Trypsin
  • bisindolylmaleimide I
  • Alanine