title: Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1.
abstract: Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Ning
authors: N
authors: Zhang
authors: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom.
authors: Sarah L
authors: http://orcid.org/0000-0003-3265-8977
authors: SL
authors: Gordon
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Maximilian J
authors: http://orcid.org/0000-0001-7850-9649
authors: MJ
authors: Fritsch
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Noor
authors: N
authors: Esoof
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: David G
authors: http://orcid.org/0000-0003-2278-2149
authors: DG
authors: Campbell
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Robert
authors: R
authors: Gourlay
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Srikannathasan
authors: S
authors: Velupillai
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Thomas
authors: http://orcid.org/0000-0002-6745-183X
authors: T
authors: Macartney
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Mark
authors: http://orcid.org/0000-0002-8337-3122
authors: M
authors: Peggie
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and.
authors: Daan M F
authors: DM
authors: van Aalten
authors: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom m.cousin@ed.ac.uk d.r.alessi@dundee.ac.uk.
authors: Michael A
authors: MA
authors: Cousin
authors: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and m.cousin@ed.ac.uk d.r.alessi@dundee.ac.uk.
authors: Dario R
authors: DR
authors: Alessi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry / Q000235:genetics; D047389:Casein Kinase I / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D050863:Synaptotagmin I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C489614:Sv2a protein, mouse; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse; D047389:Casein Kinase I
keywords: CK1; SV2A; synaptotagmin