Abstract
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, alpha/beta-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.
Highlights
Amphiphysin I is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE)
Amphiphysin I in Vivo Phosphorylation Sites that produce different sized protein products, but these do not appear to account for the amphI phosphorylated doublet, nor have they yet been detected as phosphoproteins in vivo (9 –11)
Multisite Phosphorylation of AmphI in Synaptosomes—AmphI was affinity-purified from 32P-labeled rat brain synaptosomes using a combination of three amphI-binding GST fusion proteins: ␣-adaptin appendage domain, endophilin I Src homology 3 (SH3) domain, and dynamin I-proline-rich domain (PRD)
Summary
Chemicals and Constructs—All chemicals were of analytical grade or higher. Milli-Q water was used in all experiments (Milli-Q UF PLUS, Millipore). The digested solution from each sample was removed, and tryptic peptides remaining in the gel plug were extracted using 5% formic acid and were combined with the digestion solution. In some experiments a double digestion strategy was used instead of trypsin In this case, endoproteinase Arg-C (3 g) dissolved in 50 mM ammonium bicarbonate was used to digest the amphIlow and amphIup gel bands in a microwave for 15 min on medium-high setting (the Genius, 1,200 watts, Panasonic) [29]. Peptides were sequenced from the total tryptic digests, rather than only the phosphopeptide-enriched fractions, to ensure confident protein identification. This allowed specific alternatively spliced isoforms to be distinguished and eliminated redundancy in protein names reported. The peptides were conjugated to diphtheria toxoid by the C-terminal cysteine residue that was added during peptide synthesis for that purpose
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