Craft George E, Graham Mark E, Bache Nicolai, Larsen Martin R, Robinson Phillip J
Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.
Mol Cell Proteomics. 2008 Jun;7(6):1146-61. doi: 10.1074/mcp.M700351-MCP200. Epub 2008 Mar 14.
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.
在神经末梢去极化和突触小泡内吞作用（SVE）过程中，钙调神经磷酸酶可使发动蛋白I（amphI）去磷酸化。一些amphI磷酸化位点（磷酸化位点）已通过体外研究或磷酸化蛋白质组学筛选得以确定。我们采用了包括32P追踪在内的多方面策略，以识别所有体内amphI磷酸化位点，并确定它们的相对丰度以及与SVE的潜在相关性。从32P标记的突触体中提取amphI，使用二氧化钛色谱法从蛋白水解消化物中分离磷酸肽，质谱分析揭示了13个位点：丝氨酸250、252、262、268、272、276、285、293、496、514、539和626以及苏氨酸310。这些位点分布在富含脯氨酸结构域和C端Src同源3结构域周围的两个簇中。丝氨酸262的分级磷酸化先于丝氨酸268、272、276和285的磷酸化。32P标记的amphI的离线高效液相色谱分离和二维胰蛋白酶图谱分析表明，苏氨酸310、丝氨酸293、丝氨酸285、丝氨酸272、丝氨酸276和丝氨酸268的32P掺入量最高，且对刺激最敏感。单独来看，苏氨酸310和丝氨酸293是最丰富的磷酸化位点，分别掺入了32P的16%和23%。含有丝氨酸268、丝氨酸276、丝氨酸272和丝氨酸285的多个磷酸肽含有32P的27%。获得了至少一种脯氨酸导向蛋白激酶和一种非脯氨酸导向激酶发挥作用的证据。预测由非脯氨酸导向激酶作用的四个磷酸化位点，丝氨酸626、250、252和539，32P含量低且对去极化无反应。在突触体中鉴定出至少一种可变剪接的amphI异构体被组成性磷酸化，因为它在1小时标记期内未掺入32P。还鉴定了与amphI共迁移的突触体蛋白的多个磷酸化位点，包括SGIP（Src同源3结构域生长因子受体结合蛋白2（Grb2）样（内吞蛋白）相互作用蛋白1）、AAK1、eps15R、MAP6、α/β - 内收蛋白和HCN1。结果揭示了两组amphI磷酸化位点，它们在神经末梢中要么动态周转要么组成性磷酸化，这有助于更好地理解单个amphI位点或磷酸化位点簇在突触SVE中的作用。
