title: Calcineurin selectively docks with the dynamin Ixb splice variant to regulate activity-dependent bulk endocytosis.
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.
authors: Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville 2145, New South Wales, Australia.
authors: Jing
authors: J
authors: Xue
authors: Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville 2145, New South Wales, Australia.
authors: Mark E
authors: ME
authors: Graham
authors: Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville 2145, New South Wales, Australia.
authors: Aimee E
authors: AE
authors: Novelle
authors: Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville 2145, New South Wales, Australia.
authors: Nancy
authors: N
authors: Sue
authors: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
authors: Noah
authors: N
authors: Gray
authors: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
authors: Mark A
authors: MA
authors: McNiven
authors: Membrane Biology Group, Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh EH8 9XD, United Kingdom.
authors: Karen J
authors: KJ
authors: Smillie
authors: Membrane Biology Group, Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh EH8 9XD, United Kingdom.
authors: Michael A
authors: MA
authors: Cousin
authors: Cell Signalling Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville 2145, New South Wales, Australia. Electronic address: probinson@cmri.org.au.
authors: Phillip J
authors: PJ
authors: Robinson
mesh_terms: D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000818:Animals; D001921:Brain / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism / Q000502:physiology*; D034284:Dynamin I / Q000737:chemistry* / Q000378:metabolism*; D004705:Endocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011392:Proline; D005982:Glutathione Transferase; D019703:Calcineurin; D034284:Dynamin I