Kiosses William B, Hood John, Yang Suya, Gerritsen Mary E, Cheresh David A, Alderson Nazilla, Schwartz Martin Alexander
Departments of Vascular Biology, The Scripps Research Institute, La Jolla, USA.
Circ Res. 2002 Apr 5;90(6):697-702. doi: 10.1161/01.res.0000014227.76102.5d.
PAK1 is a protein kinase downstream of the small GTPases Rac and Cdc42 that previous work has implicated in endothelial cell migration via modulation of cell contraction. The first proline-rich region of PAK that binds to an SH3 domain from the adapter protein NCK was responsible for these dominant-negative effects. To test the role of PAK in angiogenesis, we prepared a peptide in which the proline-rich region was fused to the polybasic sequence from the HIV Tat protein to facilitate entry into cells. We show that the short peptide selectively binds NCK, whereas a mutant peptide does not. Treatment of cells with the PAK peptide but not the control peptide disrupts localization of PAK. This peptide specifically inhibited endothelial cell migration and contractility similarly to full-length dominant-negative PAK. In an in vitro tube-forming assay, the PAK peptide specifically blocked formation of multicellular networks. In an in vivo chick chorioallantoic membrane assay, the PAK peptide specifically blocked angiogenesis. These results, therefore, suggest a role for PAK in angiogenesis.
PAK1是小GTP酶Rac和Cdc42下游的一种蛋白激酶，之前的研究表明它通过调节细胞收缩参与内皮细胞迁移。PAK的第一个富含脯氨酸区域与衔接蛋白NCK的SH3结构域结合，正是该区域导致了这些显性负效应。为了测试PAK在血管生成中的作用，我们制备了一种肽，其中富含脯氨酸的区域与HIV Tat蛋白的多碱性序列融合，以促进其进入细胞。我们发现，这种短肽能选择性地结合NCK，而突变肽则不能。用PAK肽而非对照肽处理细胞会破坏PAK的定位。与全长显性负性PAK类似，这种肽能特异性抑制内皮细胞迁移和收缩性。在体外成管试验中，PAK肽特异性地阻断了多细胞网络的形成。在体内鸡胚绒毛尿囊膜试验中，PAK肽也特异性地阻断了血管生成。因此，这些结果表明PAK在血管生成中发挥作用。
