Buckles Thomas C, Ohashi Yohei, Tremel Shirley, McLaughlin Stephen H, Pardon Els, Steyaert Jan, Gordon Moshe T, Williams Roger L, Falke Joseph J
Molecular Biophysics Program, Department of Biochemistry, University of Colorado, Boulder, Colorado.
Medical Research Council, Laboratory of Molecular Biology Cambridge University, Cambridge, United Kingdom.
Biophys J. 2020 Dec 1;119(11):2205-2218. doi: 10.1016/j.bpj.2020.10.028. Epub 2020 Oct 31.
VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.
VPS34复合物II（VPS34CII）是一种由脂质激酶亚基VPS34和三个调节亚基组成的386 kDa组装体，它们共同作为典型的III类磷脂酰肌醇-3-激酶（PI3K）发挥作用。当活性VPS34CII复合物停靠在内体膜的细胞质表面时，它会使其底物脂质（磷脂酰肌醇，PI）磷酸化，生成重要的信号脂质磷脂酰肌醇-3-磷酸（PI3P）。反过来，PI3P会将一系列含有PI3P特异性靶向结构域（包括FYVE、PX和PROPPINs）的信号蛋白招募到膜表面，在那里它们启动关键的细胞过程。在内吞作用和早期内体发育过程中，净VPS34CII催化的PI3P产生会被Rab5A极大地放大，Rab5A是Ras GTPase超家族的一种小G蛋白。此外，VPS34CII和Rab5A都与多种人类疾病密切相关。因此，从分子层面理解Rab5A激活脂质激酶活性的机制将在信号生物学和医学领域产生广泛影响。针对小G蛋白激活PI3K脂质激酶，已经提出了两种一般的机制模型。1）在膜招募机制中，G蛋白结合增加了膜上活性激酶的密度。2）在变构激活机制中，G蛋白通过变构触发膜结合激酶分子的比活性（周转率）增加。本研究采用体外单分子方法，以阐明在目标膜表面重构的GTP-Rab5A-VPS34CII-PI3P-PX信号通路中，GTP-Rab5A相关的VPS34CII激酶激活机制。研究结果表明，膜招募和变构机制都对膜锚定的GTP-Rab5A触发的VPS34CII激酶活性和PI3P产生的大幅增加做出了重要贡献。值得注意的是，在不存在其他激活剂的近生理条件下，膜锚定的GTP-Rab5A提供了强大的、几乎是二元的开关作用，并且是VPS34CII膜结合和PI3P产生所必需的。
