Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation.
The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change.
Morel
Etienne
E
Department of Biochemistry, University of Geneva, Sciences II, 30 Quai E. Ansermet, 1211 Geneva-4, Switzerland.
Gruenberg
Jean
J
D019943:Amino Acid Substitution; D000818:Animals; D017306:Annexin A2 / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D009418:S100 Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
C472822:ANXA2 protein, human; D017306:Annexin A2; D034741:RNA, Small Interfering; D009418:S100 Proteins; C110294:S100A1 protein; D014443:Tyrosine