The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

Megan E Arrington,Brenda Temple,Antje Schaefer,Sharon L Campbell

doi:10.1074/jbc.ra120.012915

Abstract

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 (RAC2E62K) 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 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 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 RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Highlights

related C3 botulinum toxin substrate 2 (RAC2) is a 21-kDa RAS superfamily GTPase that cycles between inactive GDP- and active GTP-bound states to regulate hematopoietic cell signaling in the immune system [1,2,3]
The GTPase-activating protein (GAP) defect in RAC2E62K likely accounts for hyperactivation of RAC2E62K in RAC2E62K/1 neutrophils and increased GTP loading observed in COS cells [2]
We find that KRASE62K is defective in p120 RASGAP–stimulated GTP hydrolysis, suggesting a conserved role of Glu62 in both guanine nucleotide exchange factor (GEF) and GAP interactions

Summary

Introduction

RAC2 is a 21-kDa RAS superfamily GTPase that cycles between inactive GDP- and active GTP-bound states to regulate hematopoietic cell signaling in the immune system [1,2,3]. RAC2 activation and cell signaling are controlled by the binding of regulatory factors and effector proteins, which associate with RAC2 via two highly dynamic regions termed “switch I” (Tyr32–Asp38) and “switch II” (Ala59–Leu67) [3]. These regions rapidly “switch” between an open GDP-bound inactive conformation and a closed GTP-bound active conformation. Disease-causing mutations in RAC GTPases are rare and include the activating RAC1P29S mutant in melanoma [13] and the inactivating RAC2D57N mutant identified in CVID [6] These mutations directly perturb intrinsic guanine nucleotide binding yet do not affect GTP hydrolysis. The GAP defect in RAC2E62K likely accounts for hyperactivation of RAC2E62K in RAC2E62K/1 neutrophils and increased GTP loading observed in COS cells [2]

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Results

Conclusion