Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin
Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin
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DOI：
10.1074/jbc.m102649200
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发表时间：
2001-07-13
影响因子：
4.8
通讯作者：
Vaughan, KT
中科院分区：
文献类型：
--
作者：
Vaughan, PS;Leszyk, JD;Vaughan, KT
以前，我们确定了dynactin作为货物受体或适配器的细胞质动力蛋白，介导的动力蛋白中间链和p150（胶合）之间的相互作用，以测试磷酸化作为一个潜在的调节机制，这种相互作用，我们分析了细胞质动力蛋白的二维凝胶分析，并检测到两个中间链的变体，其中之一是消除磷酸酶处理。重叠分析表明，p150（Glued）结合去磷酸化，但不磷酸化的中间链，然后，我们进行了纯化的细胞质动力蛋白中间链的质谱，并确定了一个单一的磷酸化胰蛋白酶片段对应的p150（Glued）结合域。片段化和保留时间分析将磷酸化位点定位到丝氨酸84。设计用于模拟去磷酸化或磷酸化中间链的定点突变体破坏转染蛋白的体外磷酸化和体内磷酸化。突变体模仿脱磷酸化形式结合p150Glued在体外和过度表达扰乱动力蛋白依赖性膜的运输。模拟磷酸化形式的突变体显示减少p150（胶合）结合在体外，并没有破坏动力蛋白介导的运输时，在体内表达。这些研究结果代表了第一次映射的中间链磷酸化位点，并表明，这种磷酸化起着重要的作用，在调节细胞质动力蛋白的结合力肌动蛋白。
Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued), To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains, We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150Glued in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.