Mixed lineage kinase 2 interacts with clathrin and influences clathrin-coated vesicle trafficking
Mixed lineage kinase 2 interacts with clathrin and influences clathrin-coated vesicle trafficking
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DOI：
10.1074/jbc.m204626200
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发表时间：
2002-09-27
影响因子：
4.8
通讯作者：
Dorow, DS
中科院分区：
文献类型：
--
作者：
Akbarzadeh, S;Ji, H;Dorow, DS
混合谱系激酶2（MLK 2）是在应激激活的Jun N-末端激酶信号转导途径中发出信号的蛋白激酶。我们使用免疫沉淀和质谱分析，以确定MLK 2结合蛋白的诱导表达的绿色荧光蛋白标记的MLK 2的细胞系。在这里，我们报告的网格蛋白作为MLK 2在培养细胞和哺乳动物大脑中的结合伴侣的鉴定。我们证明，网格蛋白结合需要一个基序（LLDMD）位于MLK 2的C端附近，这是类似于“网格蛋白盒”的网格蛋白外壳组件和辅助蛋白的网格蛋白重链的结合重要的基序。含有该基序的MLK 2的C-末端片段与网格蛋白强烈结合，并且LLDMD序列突变为LAAAD完全消除网格蛋白结合。我们从表达绿色荧光蛋白MLK 2的细胞和小鼠脑裂解物中分离网格蛋白包被的囊泡，发现MLK 2在这些囊泡中沿着网格蛋白。此外，我们证明了内源性MLK 2与来自小鼠脑裂解物的囊泡富集级分的网格蛋白重链共免疫沉淀。此外，MLK 2在培养细胞中的过表达抑制受体介导的内吞作用期间标记的转铁蛋白在再循环内体中的积累。这些发现表明MLK 2和应激信号通路在囊泡形成或运输中的网格蛋白活性位点处的作用。
Mixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to "clathrin box" motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking.