Haffner C, Jarchau T, Reinhard M, Hoppe J, Lohmann S M, Walter U
Medizinische Universitätsklinik, Klinische Forschergruppe, Würzburg, Germany.
EMBO J. 1995 Jan 3;14(1):19-27. doi: 10.1002/j.1460-2075.1995.tb06971.x.
The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks.
血管舒张刺激磷蛋白（VASP）在体外和完整细胞中是环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）依赖性蛋白激酶的底物，与肌动蛋白丝、粘着斑和动态膜区域相关。本文从人HL - 60细胞和犬MDCK细胞中克隆得到的VASP由三个不同结构域组成。中央富含脯氨酸的结构域含有一个单拷贝的GPPPPP基序和一个三倍串联重复序列，以及三个环核苷酸依赖性蛋白激酶的保守磷酸化位点。C末端结构域含有一个预测会形成α螺旋的重复混合电荷簇。纯化的人VASP的流体动力学性质以及克隆的VASP的计算分子量表明，天然蛋白是一种具有拉长结构的同四聚体。在瞬时转染的BHK21细胞中过表达的VASP主要在应力纤维、粘着斑和含F - 肌动蛋白的细胞表面突起中被检测到，而缺少C末端结构域的截短VASP不再集中在粘着斑处。这些数据表明，C末端结构域是将VASP锚定在粘着斑位点所必需的，而中央结构域则被认为介导VASP与profilin的相互作用。我们的结果为VASP的结构基础提供了证据，VASP既是cAMP和cGMP信号转导途径的靶点，又是基于肌动蛋白的细胞骨架（包括细胞骨架 - 膜界面）的组成部分，可能能够在这些网络之间交换信号。
