The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cistrans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/ cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo. Peptidyl-prolyl isomerases (PPIases)2 accelerate the cis-trans conversion of peptide bonds preceding prolyl residues, which can cause alterations in protein conformation (e.g. see Refs. 1 and 2). PPIases have been grouped into cyclophilin, FK506-binding protein, and parvulin subfamilies (see Ref. 3), for which distinct pharmacological inhibitors are available. The parvulin group is irreversibly inhibited by juglone (4). Pin1 comprises an N-terminal type IV WW domain, which determines phosphorylation-specific protein-protein interactions, and a C-terminal PPIase domain that harbors the catalytic center (see Ref. 5). Pin1 is a unique PPIase, because it preferably binds to side chain-phosphorylated S/T-P moieties in numerous proteins, including crucial cell cycle regulators or proteins that become phosphorylated immediately prior to cell division (6, 7). Isomerization of the p(S/T)-P peptide bond regulates, for instance, localization and phosphorylation status of Pin1 client proteins (see Ref. 5). Pin1 is therefore a regulator that, in concert with proline-directed kinases, phosphatases, and ubiquitin ligases, controls the cell cycle (see Refs. 5 and 8). Pin1 is possibly a cancer target gene, because its overexpression enhances transformed phenotypes induced by oncogenic Ras and Neu (see (5)). Pin1 is overexpressed in many human cancers, and its overexpression correlates with poor...