Microtubule-targeting drugs induce Bcl-2 phosphorylation and association with Pin1.
Bcl-2 is a critical suppressor of apoptosis that is overproduced in many types of cancer. Phosphorylation of the Bcl-2 protein is induced on serine residues in tumor cells arrested by microtubule-targeting drugs (paclitaxel, vincristine, nocodazole) and has been associated with inactivation of antiapoptotic function through an unknown mechanism. Comparison of a variety of pharmacological inhibitors of serine/threonine-specific protein kinases demonstrated that the cyclin-dependent kinase inhibitor, flavopiridol, selectively blocks Bcl-2 phosphorylation induced by antimicrotubule drugs. Bcl-2 could also be coimmunoprecipitated with the kinase Cdc2 in M-phase-arrested cells, suggesting that a Cdc2 may be responsible for phosphorylation of Bcl-2 in cells treated with microtubule-targeting drugs. Examination of several serine-->alanine substitution mutants of Bcl-2 suggested that serine 70 and serine 87 represent major sites of Bcl-2 phosphorylation induced in response to microtubule-targeting drugs. Both these serines are within sequence contexts suitable for proline-directed kinases such as Cdc2. Phosphorylated Bcl-2 protein was discovered to associate in M-phase-arrested cells with Pin1, a mitotic peptidyl prolyl isomerase (PPIase) known to interact with substrates of Cdc2 during mitosis. In contrast, phosphorylation of Bcl-2 induced by microtubule-targeting drugs did not alter its ability to associate with Bcl-2 (homodimerization), Bax, BAG1, or other Bcl-2-binding proteins. Since the region in Bcl-2 containing serine 70 and serine 87 represents a proline-rich loop that has been associated with autorepression of its antiapoptotic activity, the discovery of Pin1 interactions with phosphorylated Bcl-2 raises the possibility that Pin1 alters the conformation of Bcl-2 and thereby modulates its function in cells arrested with antimicrotubule drugs.
Pathan
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N
The Burnham Institute, 10901 N. Torrey Pines, La Jolla, CA 92037, USA.
Aime-Sempe
C
C
Kitada
S
S
Haldar
S
S
Reed
J C
JC
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008870:Microtubules / Q000187:drug effects* / Q000502:physiology; D008938:Mitosis / Q000502:physiology; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D005982:Glutathione Transferase; D018844:Cyclin-Dependent Kinases; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase