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

Vertommen, Didier;Rider, Mark H.;Ni, Yicheng;Waelkens, E.;Van Lint, J;et.al.
(2000) Journal of Biological Chemistry — Vol. 275, n° 26, p. 19567-19576 (2000)

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  • Vertommen, Didierorcid-logoUCLouvain
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  • Rider, Mark H.UCLouvain
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  • Ni, YichengUCLouvain
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  • Waelkens, E.
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  • Van Lint, J
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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.
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Vertommen, D., Rider, M. H., Ni, Y., Waelkens, E., Merlevede, W., Vandenheede, J. R., & Van Lint, J. (2000). Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis. Journal of Biological Chemistry, 275(26), 19567-19576. https://doi.org/10.1074/jbc.M001357200 (Original work published 2000)