Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA; R. L. Juliano Structural Bioinformatics Core Facility, University of North Carolina, Chapel Hill, North Carolina, USA.
J Biol Chem. 2020 Aug 21;295(34):12130-12142. doi: 10.1074/jbc.RA120.012915. Epub 2020 Jul 7.
The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2 Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2 is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2 As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2 retains binding to an NADPH oxidase (NOX2) subunit, p67, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2 mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.
RAS 相关的 C3 型肉毒梭菌毒素底物 2（RAC2）是 RAS 家族 GTP 酶 RHO 亚类的成员，对于正常的免疫功能是必需的。在常见可变免疫缺陷患者中，已经鉴定出 RAC2 （RAC2）中参与识别调节因子和效应物的关键开关 II 区域的激活突变。为了更好地理解突变如何使 RAC2 功能失调，我们评估了 RAC2 的结构和稳定性、鸟嘌呤核苷酸交换因子（GEF）和 GTP 酶激活蛋白（GAP）活性以及效应物结合。我们的研究结果表明，E62K 突变不会改变 RAC2 的结构或稳定性。然而，它确实改变了 GEF 的特异性，因为 DOCK GEF、DOCK2 可激活 RAC2，但 Dbl 同源 GEF、TIAM1 不能激活其亲本蛋白。我们之前的数据还表明，E62K 突变会损害 RAC2 的 GAP 活性。由于这种疾病突变也存在于 RAS GTP 酶中，我们评估了 GAP 对 KRAS 刺激的 GTP 水解作用，并观察到类似的损伤，这表明该突变在 GAP 激活中发挥保守作用。我们还研究了 E62K 突变是否改变了效应物结合，因为激活的 RAC2 结合效应物以通过效应物途径传递信号。我们发现，RAC2 仍然与 NADPH 氧化酶（NOX2）亚基 p67 以及 p21 激活激酶的 RAC 结合结构域结合，这与我们之前的发现一致。总之，我们的研究结果表明，RAC2 突变通过促进 RAC2 的过度激活、改变 GEF 的特异性以及损害 GAP 功能而保留关键的效应物相互作用，从而促进免疫功能障碍。
