Phosphorylation of mitotic proteins on the Ser/Thr-Pro motifs has been shown to play an important role in regulating mitotic progression. Pin1 is a novel essential peptidyl–prolyl isomerase (PPIase) that inhibits entry into mitosis and is also required for proper progression through mitosis, but its substrate(s) and function(s) remain to be determined. Here we report that in both human cells and Xenopus extracts, Pin1 interacts directly with a subset of mitotic phosphoproteins on phosphorylated Ser/Thr-Pro motifs in a phosphorylation-dependent and mitosis-specific manner. Many of these Pin1-binding proteins are also recognized by the monoclonal antibody MPM-2, and they include the important mitotic regulators Cdc25, Myt1, Wee1, Plk1, and Cdc27. The importance of this Pin1 interaction was tested by constructing two Pin1 active site point mutants that fail to bind a phosphorylated Ser/Thr-Pro motif in mitotic phosphoproteins. Wild-type, but not mutant, Pin1 inhibits both mitotic division in Xenopusembryos and entry into mitosis in Xenopus extracts. We have examined the interaction between Pin1 and Cdc25 in detail. Pin1 not only binds the mitotic form of Cdc25 on the phosphorylation sites important for its activity in vitro and in vivo, but it also inhibits its activity, offering one explanation for the ability of Pin1 to inhibit mitotic entry. In a separate paper, we have shown that Pin1 is a phosphorylation-dependent PPIase that can recognize specifically the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. Thus, Pin1 likely acts as a general regulator of mitotic proteins that have been phosphorylated by Cdc2 and other mitotic kinases.

All-trans retinoic acid binds to, inhibits and induces degradation of the active form of the prolyl isomerase Pin1, thereby turning off and on a variety of Pin1 substrate oncogenes and tumor suppressors, respectively. A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)—a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive—inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML–RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.