C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins Crk and CrkL and by tyrosine phosphorylation. Here, we uncovered mechanisms of C3G autoinhibition and activation. Specifically, we found that two intramolecular interactions regulate the activity of C3G. First, an autoinhibitory region (AIR) within the central domain of C3G binds to and blocks the catalytic Cdc25H domain. Second, the binding of the protein’s N-terminal domain to its Ras exchanger motif (REM) is required for its GEF activity. CrkL activated C3G by displacing the AIR/Cdc25HD interaction. Two missense mutations in the AIR found in non-Hodgkin’s lymphomas, Y554H and M555K, disrupted the autoinhibitory mechanism. Expression of C3G-Y554H or C3G-M555K in Ba/F3 pro–B cells caused constitutive activation of Rap1 and, consequently, the integrin LFA-1. Our findings suggest that sustained Rap1 activation by deregulated C3G might promote progression of lymphomas and that designing therapeutics to target C3G might treat these malignancies.

C3G 是一种鸟嘌呤核苷酸交换因子 (GEF)，它通过激活 GTPase Rap1 来调节细胞粘附和迁移。C3G 的 GEF 活性受到衔接蛋白 Crk 和 CrkL 以及酪氨酸磷酸化的刺激。在这里，我们发现了 C3G 自动抑制和激活的机制。具体而言，我们发现两种分子内相互作用调节 C3G 的活性。首先，C3G 中心域内的自抑制区 (AIR) 结合并阻断催化 Cdc25H 域。其次，蛋白质的 N 端结构域与其 Ras 交换基序 (REM) 的结合是其 GEF 活性所必需的。CrkL 通过取代 AIR/Cdc25HD 相互作用激活 C3G。在非霍奇金淋巴瘤中发现的两个 AIR 错义突变 Y554H 和 M555K 破坏了自身抑制机制。C3G-Y554H 或 C3G-M555K 在 Ba/F3 pro-B 细胞中的表达导致 Rap1 的组成型激活，从而导致整联蛋白 LFA-1。我们的研究结果表明，通过失调的 C3G 持续激活 Rap1 可能会促进淋巴瘤的进展，并且设计靶向 C3G 的疗法可能会治疗这些恶性肿瘤。