Meerschaert K, De Corte V, De Ville Y, Vandekerckhove J, Gettemans J
Flanders Interuniversity Institute for Biotechnology (V.I.B.) and Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium.
EMBO J. 1998 Oct 15;17(20):5923-32. doi: 10.1093/emboj/17.20.5923.
An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.
我们开展了一项广泛的调查，以寻找能够以类似于磷脂酰肌醇4,5-二磷酸（PI 4,5-P2）的方式调节肌动蛋白结合蛋白的化合物。为此，我们开发了一种灵敏的检测方法，该方法涉及从凝溶蛋白P-肌动蛋白复合物中释放放射性磷酸化肌动蛋白。我们发现，结构最简单的溶血磷脂，溶血磷脂酸（LPA），可使凝溶蛋白P与肌动蛋白之间的复合物解离，而其他溶血磷脂或鞘氨醇-1-磷酸则无活性。此外，LPA抑制了从血浆中纯化或作为重组蛋白的人凝溶胶蛋白、小鼠促解聚蛋白和多头绒泡菌凝溶蛋白P的F-肌动蛋白切断活性。通过凝胶过滤分析LPA对含肌动蛋白复合物的解离作用，结果表明与PI 4,5-P2不同，LPA以单体形式具有活性。我们进一步表明，LPA与这些肌动蛋白调节蛋白的结合促进了pp60（c-src）对它们的磷酸化作用。一种PI 4,5-P2结合肽可抵消LPA介导的作用，这表明LPA与这些肌动蛋白结合蛋白中的相同靶区域结合。当同时使用LPA和PI 4,5-P2时，我们发现LPA降低了PI 4,5-P2发挥活性的阈值浓度。值得注意的是，LPA促进了辛基葡糖苷通透的人血小板中凝溶胶蛋白从肌动蛋白丝末端的释放。这些结果表明，溶血磷脂酸可能作为肌动蛋白结合蛋白的细胞内调节剂。我们的发现还可以解释激动剂诱导的、并非由多磷酸肌醇介导的肌动蛋白细胞骨架变化。
