Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2 Ann Kari Grindheim 1,2 , Hanne Hollås 1 , Juan Ramirez 3 , Jaakko Saraste 1,2 , Gilles Travé 3 and Anni Vedeler 1 1 - Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway 2 - Molecular Imaging Center (MIC), University of Bergen, N-5009 Bergen, Norway 3 - Biotechnologie et Signalisation Cellulaire UMR 7242, Ecole Supérieure de Biotechnologie de Strasbourg, F-67412 Illkirch, France Correspondence to Anni Vedeler: Jonas Lies Vei 91, N-5009 Bergen, Norway. Anni.Vedeler@biomed.uib.no http://dx.doi.org/10.1016/j.jmb.2014.04.019 Edited by P. Griffin Abstract Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3′ untranslated region and β-γ-G-actin with high affinity in a Ca 2+ -independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca 2+ -dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca 2+ -dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein–lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca 2+ levels, while the Ca 2+ -dependent binding of AnxA2 to phospholipids is attenuated. © 2014 Elsevier Ltd. All rights reserved. Introduction Annexin A2 (AnxA2), a multifunctional ~ 39-kDa protein of the annexin superfamily, was originally characterised by its ability to bind negatively charged phospholipids in a Ca 2+ -dependent manner [1–5]. Structurally, AnxA2 harbours the conserved annexin core structure containing four domains and adopting the shape of a slightly curved disc with a concave and a convex surface. The N-terminal end of AnxA2, located along the concave surface of the core structure [6], folds into a structurally separate domain when bound to the EF-hand protein S100A10 [7]. However, from Pro20 (counting the first Ser as 1 throughout the text), it is likely to establish interactions with hydrophobic residues on the surface of the core structure and not with interior residues as observed in AnxA1 [8,9]. In addition to calcium and phospholipids that bind to the convex surface of the core structure [1,2], interactions of AnxA2 with, for example, G-actin [10,11] and/or F-actin [12], specific mRNAs [13], tissue plasminogen activator and plasminogen/plasmin [14] have been reported. S100A10 is an important major ligand. Two copies of AnxA2 can bind to an S100A10 homodimer and thus form an AnxA2-S100A10 hetero- tetramer. While monomeric AnxA2 is preferentially found soluble in the cytosol, formation of the hetero- tetrameric complex leads to translocation of AnxA2 to the plasma membrane or intracellular membrane IMF YJMBI-64434; No. of pages: 14; 4C: 4, 8 0022-2836/© 2014 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2014) xx, xxx–xxx Article Please cite this article as: Grindheim Ann Kari, et al, Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2, J Mol Biol (2014), http://dx.doi.org/10.1016/j.jmb.2014.04.019