Mechanism of N-Wasp Activation by Cdc42 and Phosphatidylinositol 4,5-Bisphosphate

Rohatgi, Rajat; Ho, Hsin‐Yi Henry; Kirschner, Marc W.

doi:10.1083/jcb.150.6.1299

Cited by 560 publications

(503 citation statements)

References 37 publications

(82 reference statements)

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“…The mammalian Cdc42 effector N-WASP, a regulator of actin assembly, provides a well-studied paradigm for synergism between protein and phospholipid binding (Prehoda et al, 2000;Rohatgi et al, 2000). N-WASP activation requires Cdc42 to bind in concert with PIP 2 , which acts through a polybasic domain located similarly to the BR domains of yeast Ste20, Gic1, and Gic2.…”

Section: Synergistic Protein-protein and Protein-membrane Interactionsmentioning

confidence: 99%

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

Takahashi

Pryciak

2007

MBoC

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The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP 2 -containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase-dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein-protein and protein-membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.

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Section: Synergistic Protein-protein and Protein-membrane Interactionsmentioning

confidence: 99%

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

Takahashi

Pryciak

2007

MBoC

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“…The multiple domains integrate signals that induce the following: 1) WASP recruitment to activation sites (e.g., immune synapse); and 2) conformational activation by exposure of the Cterminal VCA (verprolin/cofilin/acidic) domains that catalyze nucleation of actin filaments (8,9). WASP is found in the cytoplasm of resting cells in the inactive autoinhibited conformation in which the VCA domains are bound to the central GTPase binding domain (GBD) (10).…”

mentioning

confidence: 99%

Genotype-Proteotype Linkage in the Wiskott-Aldrich Syndrome

Lutskiy

Rosen

Remold‐O′Donnell

2005

The Journal of Immunology

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Wiskott-Aldrich syndrome (WAS) is a platelet/immunodeficiency disease arising from mutations of WAS protein (WASP), a hemopoietic cytoskeletal protein. Clinical symptoms vary widely from mild (X-linked thrombocytopenia) to life threatening. In this study, we examined the molecular effects of individual mutations by quantifying WASP in peripheral lymphocytes of 44 patients and identifying the molecular variant (collectively called proteotype). Nonpredicted proteotypes were found for 14 genotypes. These include WASP-negative lymphocytes found for five missense genotypes and WASP-positive lymphocytes for two nonsense, five frameshift, and two splice site genotypes. Missense mutations in the Ena/VASP homology 1 (EVH1) domain lead to decreased/absent WASP but normal mRNA levels, indicating that proteolysis causes the protein deficit. Because several of the EVH1 missense mutations alter WIP binding sites, the findings suggest that abrogation of WIP binding induces proteolysis. Whereas platelets of most patients were previously shown to lack WASP, WASP-positive platelets were found for two atypical patients, both of whom have mutations outside the EVH1 domain. WASP variants with alternative splicing and intact C-terminal domains were characterized for eight nonsense and frameshift genotypes. One of these, a nonsense genotype in a mild patient, supports expression of WASP lacking half of the proline-rich region. With one notable exception, genotype and proteotype were linked, indicating that a genotype-proteotype registry could be assembled to aid in predicting disease course and planning therapy for newly diagnosed infants. Knowledge of the molecular effect of mutations would aid also in identifying disease-modifying genes.

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“…Evidence suggests that the formation of filopodia is rescued in E2 and E4 cell lines after retroviral transduction with a wild-type WASP gene. It has been proposed that bradykinin acts by activating Cdc42 which, in turn, stimulates WASP/N-WASP activity and induces actin polymerization to generate filopodia (5,14,19,24). While the role of N-WASP in this signaling cascade seems to be clear, the participation of WASP has been questioned.…”

Section: Discussionmentioning

confidence: 97%

“…This process has been studied in non-hematopoietic cells and was shown to be dependent on Cdc42, a GTPase that binds to hematopoietic-cell-specific WASP (15,16), causing a conformational change that allows WASP to interact with the Arp2/3 complex and initiate actin polymerization (17)(18)(19).…”

Section: Actin Microspike Formation After Bradykinin Stimulation Is Dmentioning

confidence: 99%

Novel membrane cell projection defects in Wiskott-Aldrich syndrome B cells

Andreu

Aran²,

Fillat³

2007

Int J Mol Med

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Abstract. Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, autoimmune disorders and an increased incidence of malignancies. This complex phenotype results from mutations in the WASP gene. WASP is a key member of a protein family that links signaling pathways to actin cytoskeleton reorganization by activating Arp2/3-mediated actin polymerization. Actin polymerization defects have been extensively defined in WAS T cells and also in dendritic cells and macrophages, but few reports have concentrated on WAS B cells. In the present study, we investigated cytoskeleton abnormalities in WAS B cell lines. For the first time we report alterations in the capacity of these cells to extend filopodia in response to bradykinin stimuli and an impairment in the formation of long pseudopodia under basal conditions. Such alterations most probably result from a WASP dysfunction, given that a retroviral gene transfer of a corrected form of the WASP gene was able to rescue the abnormal phenotypes.

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Mechanism of N-Wasp Activation by Cdc42 and Phosphatidylinositol 4,5-Bisphosphate

Cited by 560 publications

References 37 publications

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

Genotype-Proteotype Linkage in the Wiskott-Aldrich Syndrome

Novel membrane cell projection defects in Wiskott-Aldrich syndrome B cells

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