title: Doxorubicin-induced activation of protein kinase D1 through caspase-mediated proteolytic cleavage: identification of two cleavage sites by microsequencing.
abstract: Recent studies have demonstrated the importance of protein kinase D (PKD) in cell proliferation and apoptosis. Here, we report that in vitro cleavage of recombinant PKD1 by caspase-3 generates two alternative active PKD fragments. N-terminal sequencing of these fragments revealed two distinct caspase-3 cleavage sites located between the acidic and pleckstrin homology (PH) domains of PKD1. Moreover, we present experimental evidence that PKD1 is an in vitro substrate for both initiator and effector caspases. During doxorubicin-induced apoptosis, a zVAD-sensitive caspase induces cleavage of PKD1 at two sites, generating fragments with the same molecular masses as those determined in vitro. The in vivo caspase-dependent generation of the PKD1 fragments correlates with PKD1 kinase activation. Our results indicate that doxorubicin-mediated apoptosis induces activation of PKD1 through a novel mechanism involving the caspase-mediated proteolysis.
authors.affiliation: Biochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Belgium.
authors.forename: Tibor
authors.initials: T
authors.lastname: Vántus
authors.forename: Didier
authors.initials: D
authors.lastname: Vertommen
authors.forename: Xavier
authors.initials: X
authors.lastname: Saelens
authors.forename: An
authors.initials: A
authors.lastname: Rykx
authors.forename: Line
authors.initials: L
authors.lastname: De Kimpe
authors.forename: Sadia
authors.initials: S
authors.lastname: Vancauwenbergh
authors.forename: Svitlana
authors.initials: S
authors.lastname: Mikhalap
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.forename: György
authors.initials: G
authors.lastname: Kéri
authors.forename: Thomas
authors.initials: T
authors.lastname: Seufferlein
authors.forename: Peter
authors.initials: P
authors.lastname: Vandenabeele
authors.forename: Mark H
authors.initials: MH
authors.lastname: Rider
authors.forename: Jackie R
authors.initials: JR
authors.lastname: Vandenheede
authors.forename: Johan
authors.initials: J
authors.lastname: Van Lint
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
chemical_list: D004317:Doxorubicin; C089212:protein kinase D; D011493:Protein Kinase C; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases