Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction.
Bcl-xL and Bcl-2 are phosphorylated in response to microtubule inhibitors, but the kinase(s) responsible and the functional significance have remained unclear. In this study, we investigated the characteristics of Bcl-xL and Bcl-2 phosphorylation in KB-3 carcinoma cells treated with vinblastine. In both asynchronous and synchronous cell cultures, Bcl-xL and Bcl-2 underwent a well-defined and coordinated cycle of phosphorylation and dephosphorylation, with a lengthy period of phosphorylation preceding apoptosis induction, and with dephosphorylation closely correlated with initiation of apoptosis. Internally, validated inhibitors of JNK, ERK, p38(MAPK), or CDK1 failed to inhibit vinblastine-induced phosphorylation of Bcl-xL or Bcl-2. In vitro, Bcl-xL and Bcl-2 were poor substrates relative to c-Jun and ATF2 for active recombinant JNK1. Both Bcl-xL and Bcl-2 were localized primarily to the mitochondrial fraction in both control and vinblastine-treated cells, indicating that phosphorylation did not promote subcellular redistribution. Bcl-xL kinase activity was demonstrated in mitochondrial extracts from vinblastine-treated, but not control, cells. These findings suggest that phosphorylation of these key antiapoptotic proteins may be catalysed by a novel or unsuspected kinase that is activated or induced in response to microtubule damage. Furthermore, the same kinase and phosphatase system may be operating in tandem on both proteins, and phosphorylation appears to maintain their antiapoptotic function, whereas dephosphorylation may trigger apoptosis. These results provide evidence for a novel signaling pathway connecting microtubule damage to apoptosis induction, and help to clarify some of the controversy concerning the role of Bcl-2 phosphorylation in microtubule inhibitor-induced apoptosis.
Du
Lihua
L
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Lyle
Christopher S
CS
Chambers
Timothy C
TC
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014747:Vinblastine / Q000494:pharmacology*; D051020:bcl-X Protein
D000972:Antineoplastic Agents, Phytogenic; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D014747:Vinblastine; D011494:Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases