Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis.
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.
Vertommen
D
D
Afdeling Biochemie, Faculteit Geneeskunde, Campus Gasthuisberg, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium.
Rider
M
M
Ni
Y
Y
Waelkens
E
E
Merlevede
W
W
Vandenheede
J R
JR
Van Lint
J
J
D000409:Alanine / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D013058:Mass Spectrometry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D011994:Recombinant Proteins; D018698:Glutamic Acid; C089212:protein kinase D; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D014357:Trypsin; C070515:bisindolylmaleimide I; D000409:Alanine