Mutations That Cause the Wiskott-Aldrich Syndrome Impair the Interaction of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein This information is current as of September 26, 2021. Donn M. Stewart, Lan Tian and David L. Nelson J Immunol 1999; 162:5019-5024; ; http://www.jimmunol.org/content/162/8/5019 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Mutations That Cause the Wiskott-Aldrich Syndrome Impair the Interaction of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein

Donn M. Stewart, Lan Tian, and David L. Nelson1

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune defi- ciency, and a proclivity toward lymphoid malignancy. Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid protein named the WAS protein (WASP). Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function. We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP

interactor. Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid Downloaded from residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant anal- ysis reveals that WASP residues 101–151 are necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS. We conclude that impaired WASP-WIP interaction may contribute to WAS. The Journal of Immunology, 1999, 162: 5019–5024.

he Wiskott-Aldrich syndrome (WAS)2 is an X-linked reces- ing proteins involved in signal transduction bind to WASP in its pro- http://www.jimmunol.org/ sive disorder characterized by eczema, thrombocytopenia line-rich segment (amino acids 310–420). These include the adaptor T with reduced mean platelet volume, immunodeficiency, and proteins Nck (26, 27) and Grb2 (28, 29), Src family kinases (29–32), a proclivity toward lymphoid malignancy (1, 2). Lymphocytes from phospholipase C␥ (29, 31), and Tec family kinases (32, 33). A cy- affected individuals have defects in activation (3, 4) and cytoskeletal toskeletal protein, proline, serine, threonine phosphatase interacting structure (5–8), and affected monocytes have impaired motility (9– protein, related to a yeast cleavage furrow protein, also associates with 11). The disease gene encodes a 502-amino acid protein, WAS pro- WASP via an SH3 domain, and this association is controlled by ty- tein (WASP), that is rich in proline (12–14). The same gene is mu- rosine phosphorylation in the SH3 domain (34). Three ABM-2 motifs tated in the disease X-linked thrombocytopenia (15–18). In vitro [(G/A/L/S)PPPPP] are found in the proline-rich region of WASP; studies have identified a number of interactions that suggest that these may represent docking sites for the cytoskeletal regulatory pro- by guest on September 26, 2021 WASP plays a role in integration of cell signaling and cytoskeletal tein profilin (23). The C terminus of WASP has domains with ho- structure and function. The N-terminal portion of WASP has two mology to the cytoskeletal proteins verprolin (VH domain; amino overlapping domains: a pleckstrin homology domain (amino acids acids 430–446) and cofilin (CH domain; amino acids 469–487) (19, 5–105), which may bind to the membrane phospholipid phosphati- 20). The VH region binds directly to actin in vitro (35). Studies of a dylinositol-4,5-biphosphate (PIP2) and cause membrane association WASP homologue expressed in neurons, neural WASP (N-WASP), of WASP (19), and a domain termed WASP homology 1 (WH1; have shown that the C-terminal fragment containing the VH and CH amino acids 47–137) (20) or Ena/VASP homology 1 (EVH1; amino domains has actin-depolymerizing activity (19, 35). N-WASP was acids 72–141) (21). The WH1/EVH1 domain may bind a proline-rich also shown to bind profilin, presumably via the ABM-2 motifs found ligand related to the actin-binding motility (ABM)-1 motif found in in its proline-rich region (35). Altogether, these studies suggest that proteins active in actin remodeling (22, 23). CDC42 and Rac are small WASP has direct activity on the actin cytoskeleton. GTPases that influence cytoskeletal structure and bind to WASP at a To investigate WASP interactions further, we performed a two- GTPase-binding motif/CDC42-interacting and -binding motif (amino hybrid screen for WASP interacting proteins. We identified a pro- acids 236–253) (20, 24, 25). Src homology 3 (SH3) domain-contain- line-rich protein with similarity to an unpublished sequence HS- PRPL2 as a specific WASP partner and, by deletion mutation analyses, identified the N terminus of WASP as the region critical Immunophysiology Section, Metabolism Branch, National Cancer Institute, National for this interaction. While our research was in progress, Ramesh et Institutes of Health, Bethesda, MD 20892 al. (36) published their findings of a similar search, which identi- Received for publication November 23, 1998. Accepted for publication January fied the same protein as a WASP binding partner and named it 28, 1999. WASP interacting protein (WIP). Our research confirms and ex- The costs of publication of this article were defrayed in part by the payment of page tends these findings, showing that WIP may have three ABM-2 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. motifs and that missense mutations in WASP that cause WAS 1 Address correspondence and reprint requests to Dr. David L. Nelson, Immunophysi- impair the WASP-WIP interaction. ology Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 4N-115, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: [email protected] Materials and Methods 2 Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; WASP, WAS Two-hybrid screen protein; PIP2, phosphatidylinositol-4,5-biphosphate; EVH1, Ena/VASP homology 1; ABM, actin-based motility; SH3, Src homology 3; N-WASP, neural WASP; VH, The interaction trap two-hybrid screen was used in these studies (37). Yeast verprolin homology; CH, cofilin homology; WIP, WASP interacting protein; GST, and vectors were obtained from Dr. Roger Brent (University of Massachu- glutathione S-methyl transferase. setts, Boston, MA). A human T-lymphotrophic virus-1-transformed T cell

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 5020 MUTATIONS THAT CAUSE WAS IMPAIR WASP-WIP INTERACTION

FIGURE 1. A, Specific interaction of WIP with WASP in yeast two-hybrid system. Yeast strains were prepared expressing various bait fusion proteins ␮

(listed to the left of the photograph) and WIP clone 9-1. A total of 10 l of yeast suspensions (1 colony in 1 ml water) were spotted onto minimal media Downloaded from with or without leucine. Growth of yeast containing the WASP bait plasmid on the leu- plate is evidence of interaction of the WIP clone 9-1 and WASP. B and C, Deletion mutant analysis of WASP-WIP interaction. Suspensions of yeast strains expressing C-terminal deletion mutants of the LexA-WASP bait protein and empty library vector or WIP clone 9-1 were spotted onto minimal media with or without leucine. The mutants retain WASP amino acids as indicated by the numbers to the left of the figures. Growth on leu- plate is evidence of interaction between WIP and the WASP mutants. B, Growth of WASP mutant 1–302 in absence of WIP indicates that this deletion mutant is able to activate transcription alone and cannot be used to study interaction. http://www.jimmunol.org/

line cDNA library for this system was purchased from Clontech (Palo Alto, Liquid culture ␤-galactosidase assay CA). The screen was performed as described (38) with additional direc- ␤ tions supplied with the cDNA library. Full-length WASP cDNA was ob- Liquid culture assay for -galactosidase activity was performed using o- ␤ tained by RT-PCR from normal PBMC mRNA and cloned into the bait nitrophenyl -D-galactopyranoside (Sigma, St. Louis, MO) as a substrate vector pEG202. Expression of full-length LexA-WASP fusion protein in as described in the instructions provided with the two-hybrid screen cDNA the yeast bait strain was confirmed by Western blot analysis using both library. The assay is a modification of a published technique (41). anti-WASP (39) and anti-LexA (Clontech) mAbs. Library plasmids were rescued from yeast clones demonstrating the interaction phenotype. Plas- In vitro WASP binding assay mid DNA from these clones was sequenced and reintroduced into yeast Glutathione S-methyl transferase (GST) fusion proteins were prepared by by guest on September 26, 2021 strains expressing various bait fusion proteins to test for specificity of in- cloning PCR products of cDNA coding regions into pGEX4T-2 (Pharma- teraction. A bait plasmid containing an open reading frame of Drosophila cia, Piscataway, NJ). Proteins were expressed using the protease-deficient bicoid protein was included with the materials obtained from Dr. R. Brent. bacterial strain Escherichia coli BL21 and purified by binding to glutathi- A bait plasmid containing an open reading frame of human insulin-like ␤ one-Sepharose as described (42). Radiolabeled WASP was prepared by in growth factor I receptor cytoplasmic domain was a gift from Dr. Bhakta vitro translation of full-length WASP cDNA (wild-type and mutants) Dey (National Cancer Institute (NCI) Metabolism Branch, Bethesda, MD). cloned in pCR2 (Invitrogen) using the TNT coupled transcription-transla- Expression of the bait proteins in the yeast strains was confirmed by West- tion system (Promega, Madison, WI) in the presence of 35S-methionine. ern blot analysis using the anti-LexA mAb. GST protein (10 ␮g) bound to glutathione-Sepharose was incubated at 4°C DNA sequence analysis for 2 h with the in vitro translated protein in PBS/1% Triton X-100, washed twice at 4°C for 30 min with the same buffer, and once more overnight. Sequencing reagents and equipment were purchased from Applied Biosys- Bound protein was analyzed by SDS-PAGE and autoradiography. tems (Foster City, CA). Fluorescent dideoxy terminator sequencing reac- tions were performed according to manufacturer’s instructions and were Results analyzed using the 377 automated sequencer and accompanying software. Several potential WASP interacting clones were identified in the RT-PCR initial screen. To test for specificity of interaction, these library Blood donors were volunteers giving informed consent under Institutional plasmids were introduced into yeast bait strains expressing LexA Review Board-approved NCI Metabolism Branch protocols. PBMC were without a fusion partner, or fused to bicoid protein, insulin-like isolated from whole blood or apheresis specimens as described (40). Total growth factor I receptor ␤ cytoplasmic domain, or WASP. Several RNA was isolated using Trizol reagent (Life Technologies, Gaithersburg, MD) according to manufacturer’s instructions. mRNA was isolated from clones showed specific interaction with WASP. One clone, 9-1, the total RNA using the Fast-Track kit (Invitrogen, Carlsbad, CA). Reverse encoded a cDNA highly similar to the proline-rich protein HS- transcription was performed using the cDNA Cycle kit (Invitrogen). PCR PRPL2 and interacted specifically with WASP (Fig. 1A). This se- was performed using AmpliTaq polymerase (Applied Biosystems). PCR quence has been deposited in GenBank with the accession no. products were purified before sequencing using the QIAquick kit (Qiagen, Chatsworth, CA). AF106062. After our work was in progress, Ramesh et al. (36) published a protein sequence similar to clone 9-1, naming it WASP Mutation analysis interacting protein (WIP). The sequence of clone 9-1 is identical to LexA-WASP C-terminal deletion mutants were prepared by cloning PCR the previously published WIP sequence (GenBank accession no. products representing portions of WASP cDNA into the bait vector AF031588) from nucleotide 680 to 1690 with the following dif- pEG202. The deletion mutants extended from amino acid 1 to 101, 151, ferences in the coding region. The sequence has a different reading 176, 201, 302, or 442. Point mutants of LexA-WASP or WASP were frame between nucleotides 1012 and 1035 (referenced to the pre- prepared using the Quick Change kit (Stratagene, La Jolla, CA). All point mutants were sequenced to confirm the presence of the mutation. All mu- viously published WIP sequence) due to CC pairs found at 1013 tant bait constructs were tested for protein expression in yeast by Western and 1036; the previously published sequence has a single C at blot analysis using the anti-LexA mAb (Clontech). 1013 and CCC at 1035. This altered reading frame results in the The Journal of Immunology 5021

FIGURE 3. WASP missense mutations interfere with WIP interaction

as detected by ␤-galactosidase production. Yeast clones expressing the Downloaded from wild-type or mutant WASP bait proteins and WIP clone 9-1 were subjected to liquid culture ␤-galactosidase assay as described in Materials and Meth- ods. Enzyme activity detected at 3 and5hisindicated as absorbance at 420 nm, normalized to a culture OD 600 nm of 1.0. Three independent assays FIGURE 2. WASP missense mutations interfere with WIP interaction of each culture were performed and the results shown as the mean (bar) and as detected by growth on leu- media. Suspensions of yeast strains express- the SD (whiskers). Impairment of ␤-galactosidase production with the http://www.jimmunol.org/ ing either empty bait vector or LexA-WASP bait proteins with missense WASP missense mutations is evidence of impaired interaction of WASP mutations (listed at the top) and either empty library vector or WIP clone with WIP. The results are representative of two experiments using different 9-1 (listed to the left) were diluted to the OD 600 nm listed to the right of clones picked from the transformation plates. the plate photographs. A total of 10 ␮l of each suspension was spotted onto minimal media with or without leucine. Poor growth of yeast strains with the WASP missense mutants R86H, Y107C, and A134T on leu- media compared with the wild-type is evidence for an impaired interaction of A number of missense mutations of WASP that cause WAS WASP with WIP. are located in the first 151 amino acids (43). Three of these were

tested for their effect on the WASP-WIP interaction in the two- by guest on September 26, 2021 hybrid system. Point mutations causing the amino acid substi- tutions R86H, Y107C, and A134T were introduced into the protein sequence 301-SASSQAPPPPPP-312 (the seven amino ac- LexA-WASP 1–201 bait vector. These mutant bait strains, ids different from the previously published sequence are under- along with the empty bait vector negative control and the wild- lined). The sequence APPPPP is an ABM-2 motif. To verify this type bait vector were transformed with empty library vector or difference, a full-length WIP PCR product was obtained from a B our WIP clone 9-1. Fig. 2 shows the growth of these various cell cDNA library using primers flanking the WIP coding se- strains on selective media. Impairment of growth of all three quence. This product was cloned and was found to have the same mutants on leucine-free media was observed, with the A134T sequence in this region as clone 9-1. In addition, RT-PCR of WIP mutant showing the most impairment. To further characterize nucleotides 810-1140 was performed using cDNA from mRNA the interaction, a liquid culture ␤-galactosidase assay was per- obtained from PBMC from three different normal volunteer do- nors. Three different amplifications were performed from each RT formed as shown in Fig. 3. The assay showed impairment in ␤ reaction to exclude mutations introduced by PCR. PCR products -galactosidase production of all three WASP mutants after 3 h were sequenced directly and showed the same sequence as our in induction media, and of A134T and R86H after 5 h. These original clone 9-1. Taken together, these results show that the WIP results are consistent with the hypothesis that these mutations sequence contains three ABM-2 motifs. In addition, a GGT to impair the interaction of WASP with WIP. The order of severity Ͼ Ͼ GCT change in codon 495 (nucleotide 1591 in AF031588) in the impairment is A134T R86H Y107C. clone 9-1 results in a conservative Gly to Ala substitution. This To further investigate and confirm the impairment of the mutant substitution was also seen in the full-length PCR product obtained WASP-WIP interactions, an in vitro binding assay was performed. from the B cell library. This may represent an allelic difference. Radiolabeled full-length WASP of wild-type or with the same sub- To identify the regions of WASP important for WIP binding, a stitution mutants as tested in the two-hybrid system were prepared series of C-terminal deletion mutants of the LexA-WASP bait pro- by in vitro translation. Fig. 4 shows binding of these WASP prep- tein were prepared and tested for WIP interaction using the clone arations to a GST-WIP fusion protein that contained amino acid 9-1 (Fig. 1B). Of these mutants, LexA-WASP 1–302 was a strong residues 416–503 of WIP, representing the minimal WASP bind- activator of transcription of the reporter genes by itself and could ing fragment identified by Anton et al. (44). The binding of wild- not be used for this analysis (Fig. 1B). The significance of this type WASP to GST-WIP 416–503 is stronger than the binding of observation with respect to the function of WASP itself is un- the WASP mutants. No clear difference between the mutants could known. WIP interacted with a WASP deletion mutant retaining be appreciated with this test. It is possible that the two-hybrid amino acids 1–201, but not with a mutant with amino acids 1–101 system is more sensitive to the strength of interactions than the in (Fig. 1B). Further analysis showed that WIP interacted with a mu- vitro test, allowing small differences in affinity to be better tant retaining amino acids 1–151 (Fig. 1C). appreciated. 5022 MUTATIONS THAT CAUSE WAS IMPAIR WASP-WIP INTERACTION

that may mediate the WASP-WIP interaction. Perhaps the phos- pholipid and protein interactions interfere or cooperate resulting in regulatory effects. The importance of the N terminus to WASP function is highlighted by the clustering of missense mutations in this region in patients with WAS (43). There is high similarity (63.6%) between amino acids 1–151 of WASP and the corresponding residues of N-WASP. In particular, the residues R86, Y107, and A134 are all conserved between the two proteins, suggesting that WIP may also interact with N- WASP. The issue of genotype-phenotype correlation in WAS/X-linked FIGURE 4. In vitro assay of WASP-WIP interaction. Radiolabeled full- thrombocytopenia is still controversial. In general, missense mu- length WASP of wild-type or with the indicated missense mutations was incubated with GST alone or GST-WIP 416–503 coupled to glutathione tations tend to cause mild disease characterized by thrombocyto- Sepharose, and the bound proteins were analyzed by SDS-PAGE and penia and perhaps mild eczema, and null mutations tend to cause autoradiography. severe disease, characterized by thrombocytopenia, severe eczema, and immune deficiency with recurrent infections (29, 43, 46). More specifically, Zhu et al. (29) show that missense mutations in exons 1–3 tend to cause mild disease, but missense mutations in

Discussion Downloaded from exon 4 (encoding amino acids 121–154) tend to produce more At time of its discovery, there was little in the WASP primary severe disease. They reported that a patient with the mutation sequence that pointed to its function. In the past 4 years, a broad A134V, in the same codon as the mutation we tested here, had spectrum of interactions have been discovered, suggesting that severe disease, yet made WASP in an EBV-transformed cell line WASP plays an important role in integrating signaling and cy- at ϳ60% of the normal level. This suggests that the mutation ex- toskeletal structure in cells of the hematopoietic lineage. WIP, a erts a deleterious effect directly, by interfering with the WASP- proline-rich protein of 503 amino acids identified by us and WIP interaction, rather than indirectly, by affecting WASP protein http://www.jimmunol.org/ Ramesh et al. (36) has been added to the growing list of WASP stability. The A134T mutation tested here was also reported to interactors. This protein, like WASP, is a proline-rich protein with have caused classical (moderate to severe) disease (14, 43); how- homologies suggesting interactions with profilin and actin. The ever, no information on the level of protein expression is available. N-terminal sequence has a region homologous to the actin-binding Mutations in codon 86 are the most common missense mutations region of verprolin (45). The sequence of our clone shows that seen in WAS and can produce severe or mild disease (43, 47). A WIP may also have three ABM-2 motifs, suggesting that WIP may mutant WASP R86C was shown to be produced in detectable function as an actin polymerization amplification subunit to amounts in an EBV-transformed cell line, suggesting that it may WASP, binding three profilin molecules in addition to any profilin

also produce its effects directly (47). The Y107C mutation was by guest on September 26, 2021 bound by WASP itself. This is similar to the effect of VASP, which reported to produce classical WAS (43) or attenuated disease (29); has six ABM-2 motifs. When bound as a tetramer to the four a patient with mild disease was observed to make low but detect- Listeria ActA ABM-1 motifs via its EVH1 domain, VASP can able levels of the mutant protein in an EBV cell line (29). The build a complex with nearly 100 profilin binding sites and so pro- mote actin-based bacterial motility (22). WIP was shown to co- argument that these missense mutations act directly is weakened precipitate with profilin, and overexpression of WIP causes an in- somewhat by the findings of MacCarthy-Morrogh et al. (48). They crease in cellular F-actin with the development of cell surface show that in freshly isolated mononuclear cells from WAS patients ceribriform projections (36). It is not known whether WASP bind- with severe disease, protein was not detectable by immunoblot ing is necessary for these phenomena to occur, or how WASP regardless of the type of mutation (missense vs null). Furthermore, affects WIP function. It was recently shown, however, that Nck they show that the amount of protein in an EBV-transformed cell can bind both WASP and WIP, possibly regulating the WASP- line may not accurately reflect the amount of protein in the circu- WIP interaction (44). lating cells of a patient. In general, patients with severe disease had We found that the N-terminal 151 amino acids of WASP are no detectable circulating WASP, and patients with mild disease important for WIP interaction. Since a number of mutations that had detectable WASP. cause WAS occur in this portion of the molecule, we tested Studies of actin-based motility in the pathogens Listeria and several of these mutations to see if they impaired the WASP- Shigella have been very fruitful in identifying cellular compo- WIP interaction. The mutation A134T strongly impaired WIP nents involved in the production of actin filaments. The actin- binding, and the Y107C mutation impaired it noticeably. Since related protein 2/3 (Arp2/3) complex, along with VASP, are the R86H mutation also impairs WIP binding, it seems that the bound by the Listeria protein ActA, which causes production of configuration necessary for the interaction has contributions an actin tail that propels the bacterium through the cytoplasm from throughout the N terminus of WASP. No data have yet (22, 49). Arp2/3 acts to produce actin filament nucleation, and been published regarding the secondary or tertiary structure of VASP, by binding profilin, promotes actin polymerization. Nei- this region of WASP, and the effects of these mutations on the ther Arp2/3 nor VASP binding alone is sufficient to produce structure cannot be predicted. In terms of function, this region bacterial motility. Recently, N-WASP has been shown to be has been shown in N-WASP to bind to the phospholipid PIP2 required for actin-based motility of intracellular Shigella (50). (19). Based on homology with VASP and the mouse homologue The Shigella bacterial protein VirG binds N-WASP directly and of Drosophila enabled (Mena), this region may also bind a li- VASP indirectly through the actin cross-linking protein vincu- gand related to the ABM-1 motif found in the Listeria ActA lin. N-WASP may provide an actin nucleation activity, analo- protein and in the cytoskeletal proteins vinculin and zyxin (21– gous to the activity of the Arp2/3 complex required for Listeria 23). The sequence of WIP from 416 to 503 that binds to WASP motility, through its ability to sever actin filaments. This activ- has several proline-rich motifs similar to the ABM-1 sequence ity, combined with the actin polymerization activity provided The Journal of Immunology 5023 by VASP-bound profilin (and possibly by N-WASP or WIP- 17. Kolluri, R., A. Shehabeldin, M. Peacocke, A. M. Lamhonwah, bound profilin) may fulfill the requirements for actin tail for- K. Teichert-Kuliszewska, S. M. Weissman, and K. A. Siminovitch. 1995. Iden- tification of WASP mutations in patients with Wiskott-Aldrich syndrome and mation. WASP and N-WASP may be seen as proteins contain- isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus. Hum. ing motifs providing both actin nucleation activity (verprolin Mol. Genet. 4:1119. homology, cofilin homology, and acidic residues region) and 18. Zhu, Q., M. Zhang, R. M. Blaese, J. M. Derry, A. Junker, U. Francke, S. H. Chen, and H. D. Ochs. 1995. The Wiskott-Aldrich syndrome and X- actin polymerization activity (intrinsic and WIP-associated pro- linked congenital thrombocytopenia are caused by mutations of the same filin binding sites). These activities may then be localized, via gene. Blood 86:3797. interactions with PIP2 and/or SH3 domain proteins, to cellular 19. Miki, H., K. Miaura, and T. Takenawa. 1996. N-WASP, a novel actin-depoly- sites targeted for assembly of actin filaments. The activities may merizing protein, regulates the cortical cytoskeletal rearrangement in a PIP-2 dependent manner downstream of tyrosine kinases. EMBO J. 15:5326. also be regulated by interaction with SH3 domain proteins, 20. Symons, M., J. M. Derry, B. Karlak, S. Jiang, V. Lemahieu, F. McCormick, CDC42, phosphorylation, and other yet to be described inter- U. Francke, and A. Abo. 1996. Wiskott-Aldrich syndrome protein, a novel ef- actions. fector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell In conclusion, we have shown for the first time that missense 84:723. 21. Gertler, F. B., K. Niebuhr, M. Reinhard, J. Wehland, and P. Soriano. 1996. Mena, mutations that cause WAS impair the interaction of the disease- a relative of VASP and drosophila enabled, is implicated in the control of mi- gene protein WASP with another protein. This protein, WIP, has crofilament dynamics. Cell 87:227. motifs suggesting that it may act as a profilin-binding partner to 22. Niebuhr, K., F. Ebel, R. Frank, M. Reinhard, E. Domann, U. D. Carl, U. Walter, WASP. The WASP-WIP interaction depends on residues through- F. B. Gertler, J.Wehland, and T. Chakraborty. 1997. A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand out the N terminus of WASP. The need to clarify the details of the for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO function of this complex region of WASP provides a direction for J. 16:5433. Downloaded from future research. 23. Purich, D. L., and F. S. Southwick. 1997. ABM-1 and ABM-2 homology se- quences: consensus docking sites for actin-based motility defined by oligoproline regions in Listeria ActA surface protein and human VASP. Biochem. Biophys. Acknowledgments Res. Comm. 231:686. 24. Aspenstrom, P., U. Lindberg, and A. Hall. 1996. Two GTPases, Cdc42 and Rac, We thank Drs. Colin Duckett, Luigi Notarangelo, and Fabio Candotti for bind directly to a protein implicated in the immunodeficiency disorder Wiskott- their helpful review of the manuscript. Aldrich syndrome. Curr. Biol. 6:70. 25. Kolluri, R., K. F. Tolias, C. L. Carpenter, F. S. 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