title: Withaferin A binds covalently to the N-terminal domain of annexin A2.
abstract: Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Ozorowski
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Ryan
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: Hartmut
authors.initials: H
authors.lastname: Luecke
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D054358:Withanolides / Q000378:metabolism*
chemical_list: D000199:Actins; D017306:Annexin A2; D012997:Solvents; D054358:Withanolides; D003545:Cysteine; C009684:withaferin A