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Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1

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Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1 2492 • The Journal of Neuroscience, February 11, 2015 • 35(6):2492–2507 Cellular/Molecular Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1 Ning Zhang,1* X Sarah L. Gordon,2* XMaximilian J. Fritsch,1* Noor Esoof,1* XDavid G. Campbell,1 Robert Gourlay,1 Srikannathasan Velupillai,1 XThomas Macartney,1 XMark Peggie,1 Daan M.F. van Aalten,1 Michael A. Cousin,2* and Dario R. Alessi1* 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom, and 2Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom Synapticvesicleprotein2A(SV2A)isaubiquitouscomponentofsynapticvesicles(SVs).IthasrolesinbothSVtraffickingandneurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phos- phorylate 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 dysfunctionalsynaptotagmin-1retrievalduringSVendocytosisbyablatingitsphospho-dependentinteractionwithSV2A,knockdownofSV2A, 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. Key words: CK1; SV2A; synaptotagmin Introduction ubiquitously expressed in the CNS, whereas SV2B is brain spe- Synaptic vesicle protein 2 (SV2A, SV2B, and SV2C) isoforms are cific with SV2C the minor isoform (Buckley and Kelly, 1985; all integral 12 transmembrane domain synaptic vesicle (SV) pro- Bajjalieh et al., 1994). teins. SV2A is the most widely distributed SV2 isoform and is SV2A knock-out mice are normal at birth but fail to grow and experience severe seizures before premature death after 3 weeks Received Oct. 14, 2014; revised Nov. 24, 2014; accepted Dec. 15, 2014. attributable to multiple neural and endocrine deficits (Crowder Author contributions: N.Z., S.L.G., M.J.F., N.E., D.M.F.v.A., M.A.C., and D.R.A. designed research; N.Z., S.L.G., et al., 1999), indicating that SV2A plays key roles in postnatal M.J.F., N.E., D.G.C., R.G., S.V., T.M., and M.P. performed research; N.Z., S.L.G., M.J.F., N.E., D.G.C., D.M.F.v.A., M.A.C., brain function. It has been proposed to function as a positive and D.R.A. analyzed data; N.Z., S.L.G., M.J.F., N.E., M.A.C., and D.R.A. wrote the paper. effector of SV exocytosis, potentially by binding calcium This work was supported by Medical Research Council (MRC) Grant G1002117 (M.A.C.) and MRC core support (D.R.A.), Wellcome Trust Senior Fellowship Grant 087590 (D.M.F.v.A.), Wellcome Trust Equipment Award Grant (Mendoza-Torreblanca et al., 2013). All SV2 isoforms possess an 094090,andAstraZeneca,Boehringer-Ingelheim,GlaxoSmithKline,Merck,JanssenPharmaceutica,andPfizer,who N-terminal cytoplasmic domain that interacts with another inte- supported the Division of Signal Transduction Therapy Unit. We thank Paul Davies for aid with the isothermal gral SV protein, synaptotagmin-1 (Syt1; Schivell et al., 1996; calorimetric-binding analysis and for the excellent technical support of the MRC Protein Phosphorylation and Ubiq- Schivell et al., 2005; Lazzell et al., 2004). Synaptotagmin-1 is the uitylation Unit (PPU) DNA Sequencing Service (coordinated by Nicholas Helps), the MRC–PPU tissue culture team 2ϩ (coordinated by Kirsten Airey and Janis Stark), the Division of Signal Transduction Therapy protein production and key Ca sensor for evoked synchronous neurotransmitter re- antibody purification teams (coordinated by Hilary McLauchlan and James Hastie), and the Centre for Advanced lease at the synapse (Brose et al., 1992; Geppert et al., 1994), and Scientific Technologies X-Ray Crystallography Facility (coordinated by Dr. Paul Fyfe). thus its efficient incorporation into newly formed SVs during *N.Z., S.L.G., M.J.F., N.E., M.A.C., and D.R.A. contributed equally to this work. endocytosis is essential to maintain the fidelity of neurotransmis- The authors declare no competing financial interests. This article is freely available online through the J Neurosci Author Open Choice option. sion. Disruption of SV2A retrieval during SV endocytosis results Correspondence should be addressed to either of the following: Michael A. Cousin, Centre for Integrative Physi- in accumulation of synaptotagmin-1 on the plasma membrane of ology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, UK, E-mail: central nerve terminals (Yao et al. 2010), suggesting that SV2A [email protected]; or Dario R. Alessi, Medical Research Council, Protein Phosphorylation and Ubiquitylation Unit, may be required for efficient packaging of this essential calcium- College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. E-mail: [email protected]. sensing protein into SVs. DOI:10.1523/JNEUROSCI.4248-14.2015 Relatively little is understood regarding how SV2 isoforms are regu- Copyright © 2015 Zhang et al. lated. Global phosphorylation site mass spectrometry studies have re- This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse,distributionandreproductioninany vealed that SV2A is potentially phosphorylated on at least 10 residues medium provided that the original work is properly attributed. within its N-terminal cytoplasmic region (http://www.phosphosite. Zhang et al. • SV2A Phosphorylation Controls Synaptotagmin Traffic J. Neurosci., February 11, 2015 • 35(6):2492–2507 • 2493 org/proteinAction.do?idϭ8509&showAllSitesϭtrue). To our (MRC–PPU), College of Life Sciences, University of Dundee, Dundee, knowledge, only one other investigation has analyzed the regula- Scotland, UK; ]. DNA for bacterial protein expression was transformed tion of SV2A by phosphorylation (Pyle et al., 2000). This study into E. coli BL21-CodonPlus (DE3)-RIL cells (Stratagene). identified an SV-associated kinase that phosphorylated the Plasmid generation. Mouse SV2A was amplified from IMAGE N-terminal cytoplasmic domain of SV2A. In gel kinase assays and EST6493509 using KOD Hot Start DNA Polymerase (Merck Millipore), cloned into pSC-b (Agilent), and sequenced to completion. This was the use of a Casein kinase 1 (CK1) inhibitor (CK1-7) suggested then ligated into the BglII NotI sites in pCMV mCerulean (mCer) N1 that this kinase was a member of the CK1 group of eukaryotic (Anggono et al., 2006). Mutations and shRNA-resistant versions were protein kinases (Pyle et al., 2000). Intriguingly, in vitro phosphor- created using the Quick Change method (Agilent) but using KOD DNA ylation of a recombinant N-terminal fragment of SV2A compris- Hot Start polymerase. SV2A–pHluorin was created in a Clontech ing the cytoplasmic domain by CK1 increased its ability to EGFP–C1 backbone by replacing EGFP with human SV2A using XhoI interact with recombinant synaptotagmin-1 approximately and AgeI restriction enzymes. The fluorescent pHluorin protein was then threefold (Pyle et al., 2000). inserted between amino acids 197 and 198 at a PclI restriction enzyme To date, no studies have determined the site(s) phosphory- site (DU number 42587). lated by CK1 isoforms within the SV2A N-terminal cytoplasmic SV2A knockdown was achieved using the published oligonucleotide domain and how phosphorylation of these residues stimulates sequence (GAATTGGCTCAGCAGTATGttcaagagaCATACTGCTGAG recruitment of synaptotagmin-1 and/or influences SV traffick- CCAATTC) against the rat sequence of SV2A that is identical to the mouse sequence (shRNA1; Dong et al. 2006). Oligonucleotides were ing. In this study, we demonstrate that CK1 and Tau–tubulin ligated into the BglII HindIII sites of pSUPER mCer (Clayton et al., protein kinase (TTBK) isoforms efficiently phosphorylate the 2010). The SYN1 promoter-driven pHluorin–rSYT1–BGH pA cassette N-terminal cytoplasmic domain of SV2A within two constella- was amplified by adding flanking BglII and SmaI restriction sites and tions, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 ligated into either vectors pSuper.Neo mCer or pSuper.Neo mCer (Ser80, Ser81, and Thr84). We demonstrate that phosphory- mSV2A shRNA1 after digestion. lation of Thr84 in Cluster-2 is key in triggering binding to Antibodies. The following antibodies were raised in sheep by the Divi- synaptotagmin-1. Crystallographic analysis revealed that the sion of Signal Transduction Therapy at the University of Dundee and phosphorylated Thr84 residue specifically bound to a pocket affinity purified against the indicated antigens: anti-SV2A (S290D, first formed by three conserved Lys residues (Lys314, Lys326, and bleed; raised against residues 1–160 of human SV2A), anti-phospho- Lys328) on the surface of
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