Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration

Abstract

IntroductionWiskott Aldrich syndrome (WAS) is a primary immunodeficiency characterized by a broad immune defect, thrombocytopenia, severe eczema, and an increased susceptibility to both autoimmunity and hematologic malignancy. 1 The disease is caused by mutations in the gene encoding the WAS protein (WASp), a key regulator of the actin cytoskeleton in hematopoietic cells. 2,3 Cytosolic WASp exists in an autoinhibited conformation promoted by constitutive binding of the EVH1 domain to WASp Interacting Protein (WIP). [4][5][6][7][8] Activation induces a conformational change exposing the VCA (Verprolin homology, Central region, Acidic domain) domain, which binds the Arp2/3 complex, 9,10 initiating actin filament nucleation and polymerization. 11 WASp activity is regulated through the combined influence of many binding partners and posttranslational modification, of which the Rho GTPase Cdc42 and tyrosine phosphorylation have particular importance. 1 Mutations resulting in complete loss of WASp expression are predictive of severe, classic WAS while missense mutations in the EVH1 domain generally result in residual low-level WASp expression and cause milder X-linked thrombocytopenia (XLT). [12][13][14][15] Despite this, there is significant clinical heterogeneity between different EVH1 missense mutations. In particular, mutations within exon 4 are associated with a more severe clinical phenotype. 14,15 A recently published multicenter analysis of the clinical outcomes of XLT patients revealed significantly increased rates of infections, autoimmunity, and malignancy resulting in a median severe-eventfree survival of only 10.2 years. 16 Therefore, further categorization and mechanistic understanding of mutations associated with more severe clinical features is important.The common mechanism of action for disease-causing EVH1 missense mutations appears to be enhanced protein degradation as a result of reduced WIP binding. Identification of the EVH1-binding surface for WIP as a long coiled surface clarified why so many different mutations could have the same effect. Based on this model, many EVH1 mutations have either been demonstrated Submitted January 13, 2012; accepted October 11, 2012. Prepublished online as Blood First Edition paper, November 15, 2012; DOI 10.1182 DOI 10. /blood-2012.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Methods MiceC57BL/6 (B6) wild-type (Charles River Laboratories) and WAS KO mice (supplied by T. Strom, Memphis, TN) were housed in specified pathogenfree conditions and used at 6-18 weeks of age. Experiments were performed under a Home Office-approved project license (held by S.B.). Immature murine BM derived dendritic cells (BMDCs) were generated by culturing in RPMI medium supplemented with 10% FCS, 100 units/mL of penicillin, and 100 g/mL of streptomycin in...

Wiskott–Aldrich syndrome protein – dynamic regulation of actin homeostasis: from activation through function and signal termination in T lymphocytes

“…One possible pathway is masking of WASp ubiquitylation sites by WIP. In support of this mechanism, we and others recently showed that WH1deleted WASp mutants are highly resistant to degradation (40,104). These truncated forms of WASp do not bind WIP but are still protected from degradation due to the absence of ubiquitylation sites.…”

“…Both WASp and N-WASp have an N-terminal WH1 domain that serves as the binding region for the WASpinteracting protein (WIP) (38). Through this constitutive interaction, WIP controls WASp activation and localization (39)(40)(41) and most importantly protects WASp from degradation (39,42). In T cells, the WH1 domain was also identified as a binding site for the Src family kinase Fyn (43), a kinase which is known to support the activity of WASp in T cells by phosphorylating its tyrosine 291 (24).…”

“…The actin cytoskeleton is crucial for an efficient immune response. As a key actin regulator, WASp controls a variety of cellular processes by mediating the formation of various membrane structures (23,40,49,(102)(103)(104). In T cells, the actin machinery enables the cells to migrate and invade inflamed tissues, where they encounter APCs such as dendritic cells, macrophages, and B cells.…”

Current and emerging treatment options for Wiskott–Aldrich syndrome

“…Mutations associated with XLT tend to cluster in exons 2-4 (within the EVH1 domain) and are usually missense mutations predicted to interfere with WIP binding to WASp [69][70][71][72]. The reduced affinity for WIP has been demonstrated to result in an increased susceptibility to degradation of the mutated WASp protein [16,73]. Mutations causing classical WAS are more evenly distributed throughout the whole WAS gene; however, they are more likely to be deletion, nonsense or premature termination causing mutations.…”

“…A total of 69% of these patients had missense mutations, the majority of which were within the WASp EVH1 domain. Increasing evidence suggests that WASp-WIP interaction has a specific role in WASp function, beyond simply protecting WASp from degradation [31,73,132]. As the majority of XLT mutations (those in the EVH1 domain) are predicted to interfere with WIP binding to WASp, this functional defect may explain the immune dysregulation seen in patients expressing reduced, but not absent levels of WASp.…”