Phosphorylation of WASp is a key regulator of activity and stability in vivo

Blundell, Michael P.; Bouma, Gerben; Metelo, Joao; Worth, Austen; Calle, Yolanda; Cowell, Lucy A.; Westerberg, Lisa S.; Moulding, Dale; Mirando, S; Kinnon, Christine; Cory, Giles O.; Jones, Gareth E.; Snapper, Scott B.; Burns, Siobhan O.; Thrasher, Adrian J.

doi:10.1073/pnas.0904346106

Cited by 54 publications

(94 citation statements)

References 42 publications

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“…46 Conversely, the inability to phosphorylate this site reduces WASp-dependent actin polymerization in vitro and in vivo, conferring a WASpdeficiency state. 46 In the present study, we have shown that despite normal serine phosphorylation, all WASp EMM and WASp dEVH1 showed reduced tyrosine phosphorylation in vivo and we hypothesize that this results from impaired WIP-mediated retention and spatial regulation at sites of actin polymerization. In addition, WASp EMM impaired the intrinsic regulation of WASp activity in vivo, as evidenced by increased total intracellular f-actin after stimulation.…”

Section: Discussionmentioning

confidence: 99%

“…44,45 Constitutive tyrosine phosphorylation at this residue enhances WASp activity in vitro, but also targets WASp for accelerated degradation, complicating studies of effect in vivo. 46 Conversely, the inability to phosphorylate this site reduces WASp-dependent actin polymerization in vitro and in vivo, conferring a WASpdeficiency state. 46 In the present study, we have shown that despite normal serine phosphorylation, all WASp EMM and WASp dEVH1 showed reduced tyrosine phosphorylation in vivo and we hypothesize that this results from impaired WIP-mediated retention and spatial regulation at sites of actin polymerization.…”

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confidence: 99%

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

Worth

Metelo

Bouma

et al. 2013

Blood

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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...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Worth

Metelo

Bouma

et al. 2013

Blood

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“…Among WASp phosphotyrosines, tyrosine 291 (Y291) of WASp was previously shown to be the major site of WASp phosphorylation and to be critical for the activation of WASp effector function in T cells, i.e., actin polymerization (1,9). The necessity of WASp phosphorylation on Y291 for its ubiquitylation was demonstrated by biochemical approaches.…”

Section: Discussionmentioning

confidence: 99%

Ubiquitylation-Dependent Negative Regulation of WASp Is Essential for Actin Cytoskeleton Dynamics

Reicher

Joseph

David

et al. 2012

Molecular and Cellular Biology

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“…This phosphorylation site is critically involved in the activation of WASp in numerous cellular responses including proliferation, phagocytosis and especially the assembly of adhesion and chemotaxis structures. 38 Finally, these murine models have been successfully used for ten years to conduct preclinical trials evaluating somatic gene therapy as an alternative to transplantation. In fact, despite the advances in their diagnosis, the prognosis of WAS patients remains poor.…”

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confidence: 99%

The contribution of mouse models to the understanding of constitutional thrombocytopenia

et al. 2016

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Article summary• Constitutional thrombocytopenias of hereditary origin have long been poorly studied due to the difficulties of obtaining invasive marrow samples.• Genetic engineering has allowed the generation of numerous mouse models for all these pathologies, providing invaluable information to understanding these diseases and serving as preclinical models to test new therapies.Constitutional thrombocytopenias result from genetic mutations affecting platelet production. These rare diseases are still underdiagnosed, especially in adults, because they remain little-known and have a highly variable expression. Autoimmune thrombocytopenia is still often wrongly diagnosed, thereby leading to the inadequate management of patients, with occasionally inappropriate splenectomy. The diagnosis of congenital thrombocytopenia relies on cytological and functional platelet analyses performed almost exclusively in specialized laboratories. Furthermore, about 50% of thrombocytopenias, associated or not with a thrombopathy, still remain of unknown origin. 1 In cases where platelet studies orient the diagnosis to a known disease, the detection of mutations in the suspected genes can C onstitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.The contribution of mouse models to the understanding of constitutional thrombocytope...

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Phosphorylation of WASp is a key regulator of activity and stability in vivo

Cited by 54 publications

References 42 publications

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

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

Ubiquitylation-Dependent Negative Regulation of WASp Is Essential for Actin Cytoskeleton Dynamics

The contribution of mouse models to the understanding of constitutional thrombocytopenia

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