Characterization of the interaction between zyxin and members of the ena/vasodilator-stimulated phosphoprotein family of proteins
Characterization of the interaction between zyxin and members of the ena/vasodilator-stimulated phosphoprotein family of proteins
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DOI：
10.1074/jbc.m001698200
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发表时间：
2000-07-21
影响因子：
4.8
通讯作者：
Grolsteyn, RM
中科院分区：
文献类型：
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作者：
Drees, B;Friederich, E;Grolsteyn, RM
Zysin含有一个富含Pro的N-末端结构域，它类似于单核细胞增生性李斯特菌Acta蛋白中的C-末端结构域。我们筛选了人凝乳素的整个氨基酸序列，发现与ActA一样，富含脯氨酸的序列是体外唯一能够与Ena/血管扩张剂刺激的磷酸蛋白(Vasp)家族成员结合的凝乳素序列。根据这些信息，我们测试了富含脯氨酸序列的自新突变体。多肽试验、免疫沉淀和线粒体表面异位表达的凝血酶变异体证实了Mena/Vasp结合的减少。通过转染实验，我们发现在体内，Zysin与Mena/Vasp的相互作用增强了细胞顶面富含肌动蛋白的结构的产生。向细胞内显微注射与第一个富含脯氨酸的齐辛序列相对应的多肽，可导致局灶性接触中的Mena/Vasp丢失。此外，这些多肽降低了胰酶作用后复制的细胞的扩散程度，我们得出结论，凝乳素和含有类似的富含Pro重复的蛋白质有助于MENA/VASP蛋白的定位。当肌动蛋白细胞骨架重组时，例如在扩散过程中，Ena/Vasp家族成员的位置似乎很重要。
Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization, We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.