Arap3,一种用于小GTPasesArf6和RhoA的双重GTPase激活蛋白(GAP),在调节广泛的生物过程中起着关键作用,包括癌细胞的侵袭和转移。已知Arap3是可以直接结合PI(3,4,5)P3的PI3K效应物,并且PI(3,4,5)P3介导的质膜募集对于其功能至关重要。然而,该蛋白识别PI(3,4,5)P3的分子机制尚不清楚。这里,使用脂质体下拉和表面等离子体共振(SPR)分析,我们发现N端第一pleckstrin同源(PH)结构域(Arap3-PH1)可以与PI(3,4,5)P3相互作用,亲和力较低,与PI(4,5)P2。要了解Arap3-PH1和磷酸肌醇(PIP)脂质如何相互作用,我们解决了apo形式的Arap3-PH1的晶体结构,并与diC4-PI(3,4,5)P3配合物。我们还通过核磁共振(NMR)光谱表征了Arap3-PH1与溶液中的diC4-PI(3,4,5)P3和diC4-PI(4,5)P2的相互作用。此外,我们发现Arap3的过表达可以在体外抑制乳腺癌细胞的侵袭,并且PH1结构域的PIPs结合能力对于该功能是必需的。
Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.