Tyrosine Phosphorylation of Villin Regulates the Organization of the Actin Cytoskeleton*
Tyrosine Phosphorylation of Villin Regulates the Organization of the Actin Cytoskeleton*
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DOI：
10.1074/jbc.c100418200
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发表时间：
2001-09
期刊：
影响因子：
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通讯作者：
L. Zhai;Peili Zhao;A. Panebra;A. Guerrerio;S. Khurana
中科院分区：
文献类型：
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作者：
L. Zhai;Peili Zhao;A. Panebra;A. Guerrerio;S. Khurana
我们以前已经表明，酪氨酸磷酸化的肌动蛋白调节蛋白绒毛是伴随着磷酸化的绒毛的重新分配和伴随的减少，在F-肌动蛋白含量的肠上皮细胞。这两个事件的时间和空间的相关性表明，绒毛蛋白的酪氨酸磷酸化可能参与微绒毛细胞骨架的重排。通过在体外重建绒毛蛋白的酪氨酸磷酸化及其与肌动蛋白的结合，分析绒毛蛋白的酪氨酸磷酸化对肌动蛋白聚合动力学的影响，研究了这一假设。全长重组人绒毛蛋白在体外磷酸化表达TKX 1感受态细胞，携带诱导型酪氨酸激酶基因。采用共沉淀法检测绒毛蛋白的肌动蛋白结合特性。绒毛蛋白的磷酸化并不改变其与肌动蛋白的化学计量比（1：2），但降低了绒毛蛋白与肌动蛋白的结合亲和力（未磷酸化的为4.4 μm，磷酸化的为0.6 μm）。使用基于芘-肌动蛋白的荧光分析，我们证明了酪氨酸磷酸化对绒毛蛋白的肌动蛋白成核有负面影响。相反，酪氨酸磷酸化增强肌动蛋白切断绒毛。电子显微镜分析显示互补的形态学变化。磷酸化抑制肌动蛋白成束，增强肌动蛋白切断绒毛的功能。总之，我们的数据表明，绒毛蛋白的酪氨酸磷酸化减少了绒毛蛋白结合肌动蛋白丝的量，抑制肌动蛋白聚合的绒毛蛋白的属性，并促进肌动蛋白解聚的功能，而不是。这些观察结果表明，酪氨酸磷酸化的作用，在调节微绒毛细胞骨架在体内的绒毛在响应特定的生理刺激。
We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitrothe tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 μm for unphosphorylatedversus 0.6 μm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.