Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.

The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys18) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys18 by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1C18D. We also evaluated Rac1C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1WT but is resistant to thiol oxidation. In addition, Rac1C18D, but not Rac1C18S, shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1WT and the redox-insensitive Rac1C18S variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys18 is a novel posttranslational modification that upregulates Rac1 activity.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO sub-class of RAS superfamily GTPases required for proper immune function.  An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency (CVID).  To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, GEF and GAP activity, and effector binding of RAC2E62K.  Our findings indicate the E62K mutation does not alter RAC2 structure or stability.  However, it does alter GEF specificity, as RAC2E62K 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 RAC2E62K.  As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting 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 RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC binding domain of p21-activated kinase (PAK), consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyper-activation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

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