Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany.
Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Schnackenburgallee 114, 22525, Hamburg, Germany.
Biochem Biophys Res Commun. 2021 Sep 3;568:110-115. doi: 10.1016/j.bbrc.2021.06.027. Epub 2021 Jun 30.
The phosphoinositides phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P] and phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P] function as second messengers and have been implicated in cancerogenesis. The signalling events downstream of PtdIns(3,4,5)P and PtdIns(3,4)P are mediated through a complex network of phosphoinositide binding effector proteins and phosphatases. In this study, we compared the phosphoinositide effector proteins AKT1, TAPP1, TAPP2, VAV1 and P-REX1 and the phosphoinositide phosphatases PTEN, SHIP1 and INPP4B for their binding affinities to PtdIns(3,4,5)P and/or PtdIns(3,4)P using Surface Plasmon Resonance. Our results demonstrate that all measured proteins except P-REX1 and VAV1 showed high affinity phosphoinositide binding with K values in the nM to sub-nM range. Within the effector proteins, AKT1 showed the highest affinity for both PtdIns(3,4,5)P and PtdIns(3,4)P. Of the phosphoinositide phosphatases PTEN displayed the highest affinity towards PtdIns(3,4,5)P and PtdIns(3,4)P. The SHIP1 mutant E452K detected in carcinoma patients had a 100-fold increased affinity to PtdIns(3,4)P but not to PtdIns(3,4,5)P compared to SHIP1 WT. Distinct mutations in phosphoinositide binding proteins like the patient-derived SHIP1 mutant may be involved in the upregulation of PI(3,4)P -mediated signalling in tumor cells due to phosphoinositide trapping. Our results add further information to the complex hierarchy of phosphoinositide binding proteins helping to elucidate their functional role in cellular signal transduction.
磷脂酰肌醇-3,4,5-三磷酸[PtdIns(3,4,5)P]和磷脂酰肌醇-3,4-二磷酸[PtdIns(3,4)P]作为第二信使，参与癌症的发生。PtdIns(3,4,5)P 和 PtdIns(3,4)P 的下游信号事件是通过复杂的磷酸肌醇结合效应蛋白和磷酸酶网络介导的。在这项研究中，我们使用表面等离子体共振比较了 AKT1、TAPP1、TAPP2、VAV1 和 P-REX1 等磷脂酰肌醇效应蛋白和 PTEN、SHIP1 和 INPP4B 等磷酸肌醇磷酸酶与 PtdIns(3,4,5)P 和/或 PtdIns(3,4)P 的结合亲和力。结果表明，除了 P-REX1 和 VAV1 之外，所有测量的蛋白质都对 PtdIns(3,4,5)P 和/或 PtdIns(3,4)P 具有高亲和力，Kd 值在纳摩尔到亚纳摩尔范围内。在效应蛋白中，AKT1 对 PtdIns(3,4,5)P 和 PtdIns(3,4)P 的亲和力最高。在磷酸肌醇磷酸酶中，PTEN 对 PtdIns(3,4,5)P 和 PtdIns(3,4)P 的亲和力最高。在癌患者中检测到的 SHIP1 突变体 E452K 与 SHIP1 WT 相比，对 PtdIns(3,4)P 的亲和力增加了 100 倍，但对 PtdIns(3,4,5)P 没有影响。磷酸肌醇结合蛋白的独特突变，如源自患者的 SHIP1 突变体，可能会由于磷酸肌醇的捕获而导致 PI(3,4)P 介导的信号转导上调，从而参与肿瘤细胞中的信号转导。我们的结果为复杂的磷酸肌醇结合蛋白层次结构提供了进一步的信息，有助于阐明它们在细胞信号转导中的功能作用。
