PDF(Sci-hub)
Abstract：
Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1\'s cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1\'s PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1\'s activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first β-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1\'s PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
摘要：
ArfGAP与Src同源3结构域，Ankyrin重复,pleckstrin同源性（PH）域1（ASAP1）是ADP-核糖基化因子（ARF）型GTPases的多域GTPase激活蛋白（GAP）。ASAP1影响整合素粘附，肌动蛋白细胞骨架，以及癌细胞的侵袭和转移。ASAP1的细胞功能取决于其高度调节和强大的ARFGAP活性，需要ASAP1的PH和ARFGAP结构域，并受磷脂酰肌醇4,5-二磷酸（PIP2）调节。PIP2刺激的GAP活性的机理基础尚未完全了解。这里,我们调查了PIP2是否控制ARF1的N端延伸与ASAP1的PH结构域的结合,从而调节其GAP活性.使用[Δ17]ARF1，缺少N末端，我们发现PIP2对ASAP1的活性影响不大。可溶性PIP2类似物，二辛酰基-PIP2（diC8PIP2），在含N末端的变体上刺激的GAP活性，[L8K]ARF1，但仅对[Δ17]ARF1的活性有轻微影响。包含ARF1（[2-17]ARF1）的残基2-17的肽抑制GAP活性，和PIP2依赖性地结合到含有PH结构域和紧邻PH结构域的第一β链的N末端的17个氨基酸长的域间接头的蛋白质。PH结构域的接头或C末端α-螺旋中的点突变降低[2-17]ARF1结合和GAP活性。降低ARF1N末端与PH结构域结合的突变也降低了ASAP1对细胞肌动蛋白重塑的影响。降低结合的ARFN末端中的突变也降低了GAP活性。我们得出结论，PIP2调节ASAP1的PH结构域与ARF1N末端的结合，可以部分调节GAP活性。
