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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 serinel threonine-specific protein kinases demonstrated that the cyclin-dependent kinase inhibitor, flavopiridol, selectively blocks Bcl-2 phosphorylation induced by antimicrotubule drugs. Bel-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 Bcl2 phosphorylation induced in response to microtubuletargeting drugs. Both these serines are within sequence contexts suitable for proline-directed kinases such as Cdc2. Phosphorylated Bel-2 protein was discovered to associate in M-phase -arrested cells with Pint, a mitotic peptidyl prolyl isomerase (PPlase) known to interact with substrates of Cdc2 during mitosis. In contrast, phosphorylation of Bcl-2 induced by microtubuletargeting drugs did not alter its ability to associate with Bel-2 (homodimerization), Bax, BAG1, or other Bel-2binding 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 pint interactions with phosphorylated Bcl-2 raises the possibility that pint alters the conformation of Bcl-2 and thereby modulates its function in cells arrested with antimicrotubule drugs.