Effect of serine phosphorylation and Ser25 phospho-mimicking mutations on nuclear localisation and ligand interactions of annexin A2.
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.
Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway; Molecular Imaging Center (MIC), University of Bergen, N-5009 Bergen, Norway.
Ann Kari
AK
Grindheim
Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway.
Hanne
H
Hollås
Biotechnologie et Signalisation Cellulaire UMR 7242, Ecole Supérieure de Biotechnologie de Strasbourg, F-67412 Illkirch, France.
Juan
J
Ramirez
Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway; Molecular Imaging Center (MIC), University of Bergen, N-5009 Bergen, Norway.
Jaakko
J
Saraste
Biotechnologie et Signalisation Cellulaire UMR 7242, Ecole Supérieure de Biotechnologie de Strasbourg, F-67412 Illkirch, France.
Gilles
G
Travé
Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway. Electronic address: Anni.Vedeler@biomed.uib.no.
Anni
A
Vedeler
D000199:Actins / Q000378:metabolism; D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D006801:Humans; D008024:Ligands; D050356:Lipid Metabolism; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D012995:Solubility
D000199:Actins; D017306:Annexin A2; D008024:Ligands; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012694:Serine; D011493:Protein Kinase C
3′ untranslated region; G-actin; mRNA; phospholipid; secondary structure