Microtubule-targeting drugs induce Bcl-2 phosphorylation and association with Pin1

Bcl-2 is a critical suppressor of apoptosis that is overproduced in many ty pes of cancer. Phosphorylation of the Bcl-2 protein is induced on serine re sidues in tumor cells arrested by microtubule-targeting drugs (paclitaxel, vincristine, nocodazole) and has been associated with inactivation of antia poptotic function through an unknown mechanism. Comparison of a variety of pharmacological inhibitors of serine/threonine-specific protein kinases dem onstrated that the cyclin-dependent kinase inhibitor, flavopiridol, selecti vely blocks Bcl-2 phosphorylation induced by antimicrotubule drugs. Bcl-2 c ould 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 severa l serine-->alanine substitution mutants of Bcl-2 suggested that serine 70 a nd serine 87 represent major sites of Bcl-2 phosphorylation induced in resp onse to microtubule-targeting drugs. Both these serines are within sequence contexts suitable for proline-directed kinases such as Cdc2, Phosphorylate d Bcl-2 protein was discovered to associate in Rn-phase-arrested cells with Pin1,a mitotic peptidyl prolyl isomerase (PPlase) known to interact with s ubstrates of Cdc2 during mitosis, In contrast, phosphorylation of Bcl-2 ind uced 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 p roline-rich loop that has been associated with autorepression of its antiap optotic activity, the discovery of Pin1 interactions with phosphorylated Bc l-2 raises the possibility that Pin1 alters the conformation of Bcl-2 and t hereby modulates its function in cells arrested with antimicrotubule drugs.