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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M700351-MCP200v1 7/6/1146    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Google Scholar Articles by Craft, G. E. Articles by Robinson, P. J. PubMed PubMed Citation Articles by Craft, G. E. Articles by Robinson, P. J. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 7:1146-1161, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

The in Vivo Phosphorylation Sites in Multiple Isoforms of Amphiphysin I from Rat Brain Nerve Terminals^*,S

George E. Craft, Mark E. Graham, Nicolai Bache, Martin R. Larsen and Phillip J. Robinson^,¶

From the Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Locked Bag 23, Wentworthville, New South Wales 2145, Australia and Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark

Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. We used a multifaceted strategy including ³²P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from ³²P-labeled synaptosomes, phosphopeptides were isolated from proteolytic digests using TiO₂ chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC separation and two-dimensional tryptic mapping of ³²P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest ³²P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the ³²P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the ³²P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of ³²P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate ³²P during the 1-h labeling period. Multiple phosphosites from amphI-co-migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, /β-adducin, and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.

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