Juglone inactivates cysteine-rich proteins required for progression through mitosis.
The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans 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.
Department of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, The Netherlands.
Claudia
C
Fila
Corina
C
Metz
Peter
P
van der Sluijs
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002453:Cell Cycle; D006224:Cricetinae; D003412:Cricetulus; D019926:Cyclin B / Q000737:chemistry; D056744:Cyclin B1; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects*; D009285:Naphthoquinones / Q000494:pharmacology*
C534700:Ccnb1 protein, mouse; D019926:Cyclin B; D056744:Cyclin B1; D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D003545:Cysteine; C005134:juglone