Barzik Melanie, Kotova Tatyana I, Higgs Henry N, Hazelwood Larnele, Hanein Dorit, Gertler Frank B, Schafer Dorothy A
Department of Biology, University of Virginia, Charlottesville, Virginia 22903, USA.
J Biol Chem. 2005 Aug 5;280(31):28653-62. doi: 10.1074/jbc.M503957200. Epub 2005 Jun 6.
Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.
Ena/VASP蛋白影响迁移细胞板状伪足和丝状伪足以及肌动蛋白彗星尾中肌动蛋白丝网络的组织。Ena/VASP蛋白控制肌动蛋白动力学的分子机制尚不清楚。我们研究了在有和没有带刺端封端蛋白的情况下，Ena/VASP蛋白如何体外调节肌动蛋白丝带刺端的肌动蛋白聚合。重组His标签的VASP在带刺端封端蛋白、异二聚体封端蛋白（CP）、CapG和凝溶胶蛋白-肌动蛋白复合物存在的情况下提高了肌动蛋白聚合速率。肌动蛋白单体结合蛋白增强了VASP保护带刺端不被CP封端的能力，这需要肌动蛋白单体结合蛋白与G-肌动蛋白和VASP相互作用。VASP EVH2结构域足以保护带刺端不被封端，并且需要EVH2内的F-肌动蛋白和G-肌动蛋白结合基序。蛋白激酶A在VASP EVH2结构域内的位点进行磷酸化调节VASP的抗封端和F-肌动蛋白成束作用。我们提出，Ena/VASP蛋白在肌动蛋白丝带刺端或其附近结合，促进肌动蛋白组装，并限制带刺端封端蛋白与之结合。
