Journal of Proteomics & Bioinformatics
Open Access
ISSN: 0974-276X
ISSN: 0974-276X
Mark E. Graham, Nicolai Bache, Sushma Rao, Nicole Honeyman, Martin R. Larsen and Phillip J. Robinson
Accepted Abstracts: J Proteomics Bioinform
At chemical synapses, synaptic transmission is propagated by depolarization of nerve terminals which causes exocytosis of synaptic vesicles (SVs) filled with neurotransmitter. Rapid, local replenishment of SVs is required to sustain neurotransmision. Synaptic vesicle endocytosis (SVE) recycles nerve terminal membrane to create new SVs via a multi-component protein machinery. This machinery consists of a network of transient protein complexes that, i) nucleate a clathrin coat on the membrane, ii) curve membrane to create a budding vesicle, iii) pinch off the coated vesicle and, finally, iv) un-coat the vesicle. Many of the known SVE proteins are directly regulated by phosphorylation. Some phosphosites are in protein-protein interaction domains and are likely to be inhibitory to SVE. We estimate that > 50 proteins are involved in SVE with hundreds of potential phosphosites. Less than a dozen phosphosites are known to be stimulus dependent in the literature. Our aim was to define the phospho-regulated SVE protein machinery. We used pull-down experiments with GST fusions of interaction domains to target the SVE machinery. Time course experiments tracked the phosphorylation status after depolarizing stimulus. Peptide labeling with iTRAQ was used to quantify relative changes in phosphorylation status. Over 150 iTRAQ labeled phosphopeptides were confidently identified in 3 or more biological experiments. Many new stimulus-sensitive phosphorylation sites were detected in SVE proteins, proteins not previously thought to be related to SVE and in protein kinases. Furthermore, monitoring the individual phosphosites during the recovery phase has lead to the discovery of complex dynamics.
Mark Graham completed his PhD in biochemistry at The University of Newcastle (Australia) in 2002 and postdoctoral studies at the Children?s Medical Research Institute, Westmead, Australia. He has established a research group focusing on the application of phosphoproteomics to the study of neuronal and cancer related cellular signaling. He has published more than 25 research articles in these areas.