Friederich E, Vancompernolle K, Louvard D, Vandekerckhove J
Laboratoire de Morphogenèse et Signalisation Cellulaire, Centre National de la Recherche Scientifique, UMR 144, Institut Curie, 26, rue d'Ulm, Paris 75248 Cedex 05 France.
J Biol Chem. 1999 Sep 17;274(38):26751-60. doi: 10.1074/jbc.274.38.26751.
Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, and severs actin in a Ca(2+)-dependent manner in vitro. Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell transfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with its importance for microfilament bundling in cells, the KKEK motif of the carboxyl-terminal F-actin-binding site is crucial for bundling in vitro. In addition, substitutions of basic residues in a second site, located in the amino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca(2+) as well as its bundling and severing activities in vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border core bundle but does not initiate its assembly by nucleation of actin filaments.
绒毛蛋白是一种存在于肠道刷状缘的肌动蛋白结合蛋白，在体外能以钙离子依赖的方式使肌动蛋白成束、形成核、加帽并切断。绒毛蛋白可诱导转染细胞中微绒毛的生长，此活性需要位于羧基末端的KKEK基序。通过结合细胞转染和生化分析，我们发现绒毛蛋白在细胞中诱导微绒毛生长的能力与其在体外使F-肌动蛋白成束的能力相关，而与其形成核的活性无关。与其对细胞中微丝成束的重要性一致，羧基末端F-肌动蛋白结合位点的KKEK基序对体外成束至关重要。此外，绒毛蛋白氨基末端第二个位点的碱性残基被取代后，会损害其在细胞中的活性，并降低其在无钙离子情况下与F-肌动蛋白的结合以及体外的成束和切断活性。总之，这些发现表明绒毛蛋白参与刷状缘核心束的组织和稳定，但不会通过肌动蛋白丝的成核来启动其组装。
