在笼型蛋白和脂筏介导的两种内吞途径中nephrin的磷酸化修饰动力学不同

Dynamically Different Phosphorylation of Nephrin in Clathrin-and Raft-mediated Endocytosis

研究肾小球裂隙膜的主要成分nephrin分子在细胞内的转运途径及不同转运途径对nephrin磷酸化的影响.分别应用笼型蛋白介导的内吞(clathrin-mediated endocytosis,CME)和脂筏介导的内吞(raft-mediated endocytosis,RME)标记物转铁蛋白和霍乱毒素B亚基对nephrin的内吞过程进行分析,并进一步应用两种内吞途径阻断物EPS15Δ和Dyn2aK44A,研究阻断nephrin的内吞途径对其磷酸化水平的影响.结果显示,nephrin通过笼型蛋白和脂筏介导的两种内吞途径以不同速率进行内吞;与Src酪氨酸激酶家族成员Fyn共表达时,细胞内nephrin酪氨酸磷酸化被增强,而在Src家族激酶抑制剂PP2的作用下,nephrin酪氨酸磷酸化被减弱,表明nephrin的磷酸化过程是Fyn依赖的;内吞20min时,笼型蛋白介导的内吞途径的特异性阻断物EPS15Δ降低了nephrin磷酸化水平、笼型蛋白和脂筏介导的内吞途径的通用抑制剂Dyn2aK44A则增加了nephrin的磷酸化水平,综上结果表明:单独阻断脂筏介导的内吞可引起nephrin的磷酸化水平增加,脂筏介导的内吞对nephrin磷酸化过程起下调作用.

To study the involvement of nephrin phosphorylation in the endocytotic pathway, transferrin and cholera toxin subunit B （CTxB） were used as markers to study the clathrin-mediated endocytosis （CME） and raft-mediated endoeytosis （RME）. Inhibition of endocytosis by two inhibitors Epsl5A and Dyn2aK44A have different effects of CME and with Fyn increased nephrin phosphorylation, RME on the phosphorylation of nephrin. Co-expression whereas Src kinase inhibitor PP2 reduced nephrin phosphorylation. Therefore nephrin was suggested to be phosphorylated in a Fyn-dependent fashion. As a specific inhibitor of CME, EPSI5A only slightly decreased the phosphorylation of nephrin 20 min after the initiation of endocytosis; while the CME and RME general inhibitor Dyn2aK44A significantly increased the phosphorylation of nephrin. Our data have suggested that the inhibition of RME might abolish the down-regulation of nephrin phosphorylation and increased its phosphorylation level.