Extracellular phoshorylation of MAP1B and its role in synapse formation

MAP1B 的细胞外磷酸化及其在突触形成中的作用

• 批准号: 07458209
• 负责人: KURODA Yoichiro
• 金额: $ 4.67万
• 依托单位: Tokyo Metropolitan Institute for Neuroscience
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458209/
• 关键词: MAP1B; phoshorylation; ATP; K-252b; プロテオグリカン; マススペクトル; 細胞外ドメイン; シナプス可塑性; 蛋白質リン酸化; エクト・プロデインキナーゼ

Synapse formation between cultured rat cortical neurons is inhibited by the continuous application of K-252b, an ecto-protein kinase inhibitor, which cannot permeate the cell membrane. Application of K-252b also inhibited the phosphorylation of the extracellular domains of membrane proteins by ecto-protein kinase. Therefore, the phosphorylation of these membrane proteins may play important roles in synapse formation. To identify membrane proteins which may be involved in synapse formation, [gamma-^<33>P] ATP was applied to cultured cells for brief periods to phosphorylate their extracellular domains. The phosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. Some of these bands were immediately phosphorylated, and this phosphorylation was suppressed by addition of K-252b to the medium. We examined the partial amino acid sequences of these substrates. Phosphorylated bands were cut from the gel and digested with lysyl endopeptidase. Peptide fragments were separated by capillary HPLC and analyzed by mass spectrometry. The band with the highest molecular weight, whose phosphorylation was strongly inhibited by K-252b, was identified as microtubule-associated protein (MAP) 1b. These results suggest the phosphorylation of extracellular domains of MAP1b is involved in synapse formation between cortical neurons.

培养的大鼠皮层神经元之间的突触形成被抑制的连续应用K-252 b，一种胞外蛋白激酶抑制剂，不能渗透细胞膜。K-252 b的应用也抑制了胞外蛋白激酶对膜蛋白胞外结构域的磷酸化。因此，这些膜蛋白的磷酸化可能在突触形成中起重要作用。为了鉴定可能参与突触形成的膜蛋白，将[γ-β-<33>P] ATP短暂地应用于培养的细胞以磷酸化它们的细胞外结构域。用SDS-聚丙烯酰胺凝胶电泳分离磷酸化蛋白，放射自显影检测磷酸化蛋白。这些条带中的一些立即被磷酸化，并且通过向培养基中添加K-252 b来抑制这种磷酸化。我们研究了这些底物的部分氨基酸序列。从凝胶上切下磷酸化条带并用赖氨酰内肽酶消化。通过毛细管HPLC分离肽片段并通过质谱分析。分子量最大的条带被鉴定为微管相关蛋白（MAP）1b，其磷酸化被K-252 b强烈抑制。这些结果表明MAP 1b胞外区的磷酸化参与了皮层神经元之间突触的形成。

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