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  • Tyr phosphorylation of AnxA was first reported to be involve

    2021-09-07

    Tyr23 phosphorylation of AnxA2 was first reported to be involved in endocytosis, particularly in the internalisation of the insulin receptor [20]. Phosphorylated Tyr23 seems to be necessary for the stable association of AnxA2 with endosomes, and for their early-stage transport [14]. The role of AnxA2-Tyr23 phosphorylation during regulated secretion has not been studied. Here, using phosphodead and phosphomimetic mutants of AnxA2-Tyr23 generated by site-directed mutagenesis and a combination of immunocytochemical and biochemical approaches, we investigated the functional importance of Tyr23 phosphorylation during exocytosis in chromaffin cells. Our results suggest a model in which phosphorylation/dephosphorylation of AnxA2-Tyr23 regulates both lipid microdomains formation and 5 alpha reductase inhibitor filament bundling at exocytotic sites, thereby contributing to the fine tuning of regulated exocytosis.
    Materials & methods
    Results
    Discussion During neuroendocrine secretion, AnxA2 has been shown to participate together with the actin cytoskeleton in the formation of lipid microdomains required for docking and fusion of secretory granules with the plasma membrane. [28,31,34]. The N-terminal domain of AnxA2 contains many phosphorylation sites that modulate the three-dimensional structure of the protein and regulate its affinity for different partners [12,13,37]. In particular, phosphorylation of Tyr23 has been described to modify AnxA2 binding to membranes and actin [38], two major players of the exocytotic machinery [31,34]. Yet, until recently the importance of AnxA2-Tyr23 phosphorylation in the context of exocytosis has never been studied. The present report describes that AnxA2 is phosphorylated on Tyr23 during an early step of the secretory process and provides further evidence that this phosphorylation step is critical for exocytosis. Expression of AnxA2-Tyr23 phosphomimetic and phosphodead mutants in chromaffin cell indicate that a Tyr23 phosphorylation/dephosphorylation cycle is required for both the formation of lipid domains and the docking of secretory granules to the plasma membrane. We propose that AnxA2-Tyr23 is first phosphorylated to promote lipid domain formation required for secretory granule recruitment and subsequently dephosphorylated to facilitate the formation of actin bundles that stably anchor secretory granules to the plasma membrane. On the other hand, phosphorylation/dephosphorylation cycle of the AnxA2-Ser-25 has also been involved in chromaffin cells secretion [8] and phosphorylation of AnxA2-Ser11 by PKC induces the dissociation of AnxA2 tetramer [4]. In the light of these previous studies and on our results concerning the role of phosphorylation/dephosphorylation cycle of Tyr23 during exocytosis, we propose the following sequence of events depicted in Fig. 7. Upon cell stimulation, AnxA2 translocates to the plasma membrane and is phosphorylated on Tyr23 to organize lipid microdomains required for the sites of exocytosis. Tyr23 is then dephosphorylated to activate the actin bundling activity and form an actin network that stably anchors docked granules and Ser25 is phosphorylated by PKC for the late fusion step. Finally, phosphorylation on Ser11 triggers the dissociation of the AnxA2 tetramer, the relocation of AnxA2 monomer into the cytoplasm and ends the exocytotic process. Thus, the fine-tuning of AnxA2 functions in the exocytotic machinery is likely to be mediated by the coordinated modification of its three main phosphorylation sites.
    Conclusion
    Conflict of interest
    Transparency document
    Acknowledgements We are grateful to V. Gerke for generous gift of AnxA2-GFP. We thank the municipal slaughterhouse of Haguenau (France) to provide bovine adrenal glands. We acknowledge N. Grant for helpful comments and critical reading of the manuscript, B Lorber for dynamic light scattering to control liposome size. This work was supported by CNRS, Université de Strasbourg, Inserm and by a grant from Fondation pour la Recherche Médicale to N.V.