Calcineurin selectively docks with the dynamin Ixb splice variant to regulate activity-dependent bulk endocytosis

Jing Xue, Mark E. Graham, Aimee E. Novelle, Nancy Sue, Noah Gray, Mark A Mc Niven, Karen J. Smillie, Michael A. Cousin, Phillip J. Robinson

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26 Scopus citations

Abstract

Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca 2+ influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.

Original languageEnglish (US)
Pages (from-to)30295-30303
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number35
DOIs
StatePublished - Sep 2 2011

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ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Xue, J., Graham, M. E., Novelle, A. E., Sue, N., Gray, N., Mc Niven, M. A., Smillie, K. J., Cousin, M. A., & Robinson, P. J. (2011). Calcineurin selectively docks with the dynamin Ixb splice variant to regulate activity-dependent bulk endocytosis. Journal of Biological Chemistry, 286(35), 30295-30303. https://doi.org/10.1074/jbc.M111.273110