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Title: Role of Tyrosine Phosphorylation of Synaptophysin in the synaptic vesicle lifecycle
Author: Johnson, Alexander James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2012
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Synaptophysin (Syp) is a major integral synaptic vesicle (SV) protein; there are 31 copies of Syp per vesicle, which totals up to 10% of the total SV protein content. Despite being the major SV protein, little is known about the interaction partners of Syp and as a result there has been no clear role attributed to it. One key feature of Syp is that its cytoplasmic C-terminus contains 10 pentapeptide repeats, nine of which are initiated by a tyrosine residue. Syp is the major tyrosine phospho-protein on SVs. The kinase thought to phosphorylate Syp in vivo is the ubiquitously expressed non-receptor kinase C-Src. There are two splice variants of C-Src, N1- and N2-Src, which are only expressed in neuronal tissues. Although the 3 Srcs are structurally similar, they differ by a small insert of amino acids into their SH3 domains (the N-Src loop). Examination of the amino acid sequence of the cytosolic C-terminus of Syp revealed a putative type one SH3 domain interaction motif. A screen using SH3 domains of synaptic proteins as bait in GST-pull downs from nerve terminal lysate allowed an inventory of potential interaction partners of Syp to be created. Reciprocal experiments using the C-terminal of Syp as bait confirmed many of these interactions. Single point mutations of the SH3 interaction motif on Syp highlighted that syndapin and C-Src bound to Syp via this motif. These binding mutants were inserted in Syp superecliptic synaptophluorin (SypHy) to determine the functional consequences of these interactions. These mutants did not affect the trafficking of Syp when expressed in cortical neurons derived from Syp knockout mice. However, the SH3 interaction motif was fundamental for the retrieval of VAMP (vesicle associated membrane protein) when expressed in Syp knockout cultures. Importantly, this role is not mediated through a direct interaction with VAMP with the SH3 interaction motif implicating either syndapin, C-Src or both in Syp-dependent VAMP retrieval. The 3 different Srcs had different methods of interaction with Syp, and in vitro protein kinase assays the ability of the three Src splice variants to phosphorylate Syp was assessed. Key differences in both speed and efficiency of Syp phosphorylation was observed for the different Src splice variants. Mutagenesis of either all 9 tyrosine residues, only previously identified sites resulted in changes in Syp interactions in GST-pull down assays from nerve terminal lysates. To investigate the role of Syp phosphorylation in the SV lifecycle, the tyrosine pentapeptide repeats were truncated from the C-terminal of Syp in both a mCerulean tagged Syp and SypHy. The experiments showed that these potential tyrosine phosphorylation sites were not involved in the trafficking of Syp but key in the retrieval of VAMP from the plasma membrane during the SV lifecycle. I have indentified an SH3 interaction motif on the C-terminal of Syp that is critical in forming a complex of proteins that are responsible for the retrieval of VAMP during the SV lifecycle. Further experiments have shown that this key interaction is potentially phosphorylation dependent. My preliminary mass spectrometry analysis has provided a catalogue of proteins that can potentially interact with Syp, identifying proteins that may bind to either the Syp C-terminus SH3 interaction motif or to other regions in a phosphorylation dependent manner. This has provided a list of potential candidate proteins for the VAMP retrieval complex.
Supervisor: Cousin, Mike Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Tyrosine Phosphorylation ; Synaptophysin ; synaptic vesicle lifecycle