Extracellular phoshorylation of MAP1B and its role in synapse formation

培养的大鼠皮质神经元之间的突触形成被持续应用K-252b抑制，K-252b是一种不能渗透到细胞膜的胞外蛋白激酶抑制剂。K-252b的应用也抑制了胞外蛋白激酶对膜蛋白胞外区的磷酸化。因此，这些膜蛋白的磷酸化可能在突触形成中起重要作用。为了确定可能参与突触形成的膜蛋白，[Gamma-^&lt；33&gt；P]ATP被短暂地应用于培养细胞以磷酸化它们的胞外区。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离磷酸化蛋白，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，这种磷酸化被K-252b抑制。我们研究了这些底物的部分氨基酸序列。从凝胶中切下磷酸化条带，用赖氨酰内肽酶消化。用毛细管高效液相色谱分离多肽片段，并用质谱仪进行分析。被K-252b强烈抑制其磷酸化的分子量最高的条带被鉴定为微管相关蛋白(MAP)1b。这些结果表明，MAP1b胞外区的磷酸化参与了皮质神经元之间突触的形成。

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.