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.

TTBK2 and primary cilia are essential for the connectivity and survival of cerebellar Purkinje neurons

“…TTBK2 has also been shown to phosphorylate β-Tubulin as well as microtubule associated proteins TAU and MAP2 through in vitro assays (Takahashi et al, 1995;Tomizawa et al, 2001), pointing to roles for TTBK2 in the regulation of the microtubule cytoskeleton beyond the cilium. In addition, TTBK2 and the closely related kinase TTBK1 both phosphorylate SV2A in vitro, a component of synaptic vesicles important for the retrieval of the membrane trafficking protein Synaptotagmin 1 during the endocytosis of synaptic vesicles (Zhang et al, 2015). While our data showing that the Ttbk2 mutant phenotypes strongly overlap with those of other ciliary genes, such as Ift88, we can not exclude the possibility that other roles of TTBK2 specifically within the brain also contribute to the degenerative phenotypes.…”

Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway

“…For those SV proteins whose levels were reduced following 24 h cycloheximide treatment, we next evaluated whether their degradation was influenced by neuronal activity. Neurons were again treated for 24 h with DMSO or cycloheximide, this time in the presence of pharmacological agents that either block neuronal activity (the Na ϩ channel blocker TTX, the NMDA receptor blocker APV, and the AMPA receptor blocker CNQX), or increase activity (the GABA receptor antagonist bicuculline and the voltage-gated K ϩ channel blocker 4-aminopyridine; bic/4AP) (Ehlers, 2003;Tauskela et al, 2008;Wijayatunge et al, 2014). The efficacy of these agents was verified in 14 DIV neurons by recording and quantifying the frequency of spontaneous EPSCs before and after drug application.…”

“…These processes depend upon SV-associated proteins whose proper conformation and function are of paramount importance to neuronal health (Bezprozvanny and Hiesinger, 2013;Wang et al, 2013). Indeed, the buildup of damaged or misfolded SV proteins can lead to synaptic dysfunction and neurodegeneration (Garden and La Spada, 2008;Scott et al, 2010;Uytterhoeven et al, 2011).…”

“…GTP-bound Rabs in turn recruit effectors that catalyze downstream trafficking events (Stenmark, 2009). Among the Ͼ60 Rabs in mammalian brain, at least 30 are reported to associate with SV pools (Takamori et al, 2006;Pavlos and Jahn, 2011), but only a few of these have well-established roles in the SV cycle (Schlüter et al, 1999(Schlüter et al, , 2004Mahoney et al, 2006;Yu et al, 2008;Pavlos et al, 2010;Pavlos and Jahn, 2011). Several other Rabs are implicated in the endosomal sorting of SV proteins (Wucherpfennig et al, 2003;Rizzoli et al, 2006;Takamori et al, 2006;Hoopmann et al, 2010;Uytterhoeven et al, 2011).…”

“…Lysates from Rab-expressing neurons were collected and immunoblotted for SV2 and VAMP2, and the fold change in protein degradation calculated for each Rab. Rab3, an SV-associated Rab with a wellcharacterized role in SV exocytosis (Yu et al, 2008;Pavlos and Jahn, 2011), served as our control in these studies as its overexpression did not alter SV protein degradation compared with untransduced neurons (Fig. 4 F, G).…”