Doxorubicin-induced activation of protein kinase D1 through caspase-mediated proteolytic cleavage: identification of two cleavage sites by microsequencing.
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.
Vántus
Tibor
T
Biochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Belgium.
Vertommen
Didier
D
Saelens
Xavier
X
Rykx
An
A
De Kimpe
Line
L
Vancauwenbergh
Sadia
S
Mikhalap
Svitlana
S
Waelkens
Etienne
E
Kéri
György
G
Seufferlein
Thomas
T
Vandenabeele
Peter
P
Rider
Mark H
MH
Vandenheede
Jackie R
JR
Van Lint
Johan
J
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
D004317:Doxorubicin; C089212:protein kinase D; D011493:Protein Kinase C; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases