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Mutations That Cause the Wiskott-Aldrich Syndrome Impair the Interaction of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein

Immunophysiology Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

	Abstract

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Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, 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 residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant analysis 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.

	Introduction

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The Wiskott-Aldrich syndrome (WAS)² is an X-linked recessive disorder characterized by eczema, thrombocytopenia with reduced mean platelet volume, immunodeficiency, and a proclivity toward lymphoid malignancy (1, 2). Lymphocytes from affected individuals have defects in activation (3, 4) and cytoskeletal structure (5, 6, 7, 8), and affected monocytes have impaired motility (9, 10, 11). The disease gene encodes a 502-amino acid protein, WAS protein (WASP), that is rich in proline (12, 13, 14). The same gene is mutated in the disease X-linked thrombocytopenia (15, 16, 17, 18). In vitro studies have identified a number of interactions that suggest that WASP plays a role in integration of cell signaling and cytoskeletal structure and function. The N-terminal portion of WASP has two overlapping domains: a pleckstrin homology domain (amino acids 5–105), which may bind to the membrane phospholipid phosphatidylinositol-4,5-biphosphate (PIP2) and cause membrane association of WASP (19), and a domain termed WASP homology 1 (WH1; amino acids 47–137) (20) or Ena/VASP homology 1 (EVH1; amino acids 72–141) (21). The WH1/EVH1 domain may bind a proline-rich ligand related to the actin-binding motility (ABM)-1 motif found in proteins active in actin remodeling (22, 23). CDC42 and Rac are small GTPases that influence cytoskeletal structure and bind to WASP at a GTPase-binding motif/CDC42-interacting and -binding motif (amino acids 236–253) (20, 24, 25). Src homology 3 (SH3) domain-containing proteins involved in signal transduction bind to WASP in its proline-rich segment (amino acids 310–420). These include the adaptor proteins Nck (26, 27) and Grb2 (28, 29), Src family kinases (29, 30, 31, 32), phospholipase C (29, 31), and Tec family kinases (32, 33). A cytoskeletal protein, proline, serine, threonine phosphatase interacting protein, related to a yeast cleavage furrow protein, also associates with WASP via an SH3 domain, and this association is controlled by tyrosine phosphorylation in the SH3 domain (34). Three ABM-2 motifs [(G/A/L/S)PPPPP] are found in the proline-rich region of WASP; these may represent docking sites for the cytoskeletal regulatory protein profilin (23). The C terminus of WASP has domains with homology to the cytoskeletal proteins verprolin (VH domain; amino acids 430–446) and cofilin (CH domain; amino acids 469–487) (19, 20). The VH region binds directly to actin in vitro (35). Studies of a WASP homologue expressed in neurons, neural WASP (N-WASP), have shown that the C-terminal fragment containing the VH and CH domains has actin-depolymerizing activity (19, 35). N-WASP was also shown to bind profilin, presumably via the ABM-2 motifs found in its proline-rich region (35). Altogether, these studies suggest that WASP has direct activity on the actin cytoskeleton.

To investigate WASP interactions further, we performed a two-hybrid screen for WASP interacting proteins. We identified a proline-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 for this interaction. While our research was in progress, Ramesh et al. (36) published their findings of a similar search, which identified the same protein as a WASP binding partner and named it WASP interacting protein (WIP). Our research confirms and extends these findings, showing that WIP may have three ABM-2 motifs and that missense mutations in WASP that cause WAS impair the WASP-WIP interaction.

	Materials and Methods

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Two-hybrid screen

The interaction trap two-hybrid screen was used in these studies (37). Yeast and vectors were obtained from Dr. Roger Brent (University of Massachusetts, Boston, MA). A human T-lymphotrophic virus-1-transformed T cell line cDNA library for this system was purchased from Clontech (Palo Alto, CA). The screen was performed as described (38) with additional directions supplied with the cDNA library. Full-length WASP cDNA was obtained by RT-PCR from normal PBMC mRNA and cloned into the bait vector pEG202. Expression of full-length LexA-WASP fusion protein in the yeast bait strain was confirmed by Western blot analysis using both anti-WASP (39) and anti-LexA (Clontech) mAbs. Library plasmids were rescued from yeast clones demonstrating the interaction phenotype. Plasmid DNA from these clones was sequenced and reintroduced into yeast strains expressing various bait fusion proteins to test for specificity of interaction. A bait plasmid containing an open reading frame of Drosophila bicoid protein was included with the materials obtained from Dr. R. Brent. A bait plasmid containing an open reading frame of human insulin-like growth factor I receptor ß cytoplasmic domain was a gift from Dr. Bhakta Dey (National Cancer Institute (NCI) Metabolism Branch, Bethesda, MD). Expression of the bait proteins in the yeast strains was confirmed by Western blot analysis using the anti-LexA mAb.

DNA sequence analysis

Sequencing reagents and equipment were purchased from Applied Biosystems (Foster City, CA). Fluorescent dideoxy terminator sequencing reactions were performed according to manufacturer’s instructions and were analyzed using the 377 automated sequencer and accompanying software.

RT-PCR

Blood donors were volunteers giving informed consent under Institutional Review Board-approved NCI Metabolism Branch protocols. PBMC were isolated from whole blood or apheresis specimens as described (40). Total RNA was isolated using Trizol reagent (Life Technologies, Gaithersburg, MD) according to manufacturer’s instructions. mRNA was isolated from the total RNA using the Fast-Track kit (Invitrogen, Carlsbad, CA). Reverse transcription was performed using the cDNA Cycle kit (Invitrogen). PCR was performed using AmpliTaq polymerase (Applied Biosystems). PCR products were purified before sequencing using the QIAquick kit (Qiagen, Chatsworth, CA).

Mutation analysis

LexA-WASP C-terminal deletion mutants were prepared by cloning PCR products representing portions of WASP cDNA into the bait vector pEG202. The deletion mutants extended from amino acid 1 to 101, 151, 176, 201, 302, or 442. Point mutants of LexA-WASP or WASP were prepared using the Quick Change kit (Stratagene, La Jolla, CA). All point mutants were sequenced to confirm the presence of the mutation. All mutant bait constructs were tested for protein expression in yeast by Western blot analysis using the anti-LexA mAb (Clontech).

Liquid culture ß-galactosidase assay

Liquid culture assay for ß-galactosidase activity was performed using o-nitrophenyl ß-D-galactopyranoside (Sigma, St. Louis, MO) as a substrate as described in the instructions provided with the two-hybrid screen cDNA library. The assay is a modification of a published technique (41).

In vitro WASP binding assay

Glutathione S-methyl transferase (GST) fusion proteins were prepared by cloning PCR products of cDNA coding regions into pGEX4T-2 (Pharmacia, Piscataway, NJ). Proteins were expressed using the protease-deficient bacterial strain Escherichia coli BL21 and purified by binding to glutathione-Sepharose as described (42). Radiolabeled WASP was prepared by in vitro translation of full-length WASP cDNA (wild-type and mutants) cloned in pCR2 (Invitrogen) using the TNT coupled transcription-translation system (Promega, Madison, WI) in the presence of ³⁵S-methionine. GST protein (10 µg) bound to glutathione-Sepharose was incubated at 4°C 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. Bound protein was analyzed by SDS-PAGE and autoradiography.

	Results

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Several potential WASP interacting clones were identified in the initial screen. To test for specificity of interaction, these library plasmids were introduced into yeast bait strains expressing LexA without a fusion partner, or fused to bicoid protein, insulin-like growth factor I receptor ß cytoplasmic domain, or WASP. Several clones showed specific interaction with WASP. One clone, 9-1, encoded a cDNA highly similar to the proline-rich protein HS-PRPL2 and interacted specifically with WASP (Fig. 1A). This sequence has been deposited in GenBank with the accession no. AF106062. After our work was in progress, Ramesh et al. (36) published a protein sequence similar to clone 9-1, naming it WASP interacting protein (WIP). The sequence of clone 9-1 is identical to the previously published WIP sequence (GenBank accession no. AF031588) from nucleotide 680 to 1690 with the following differences in the coding region. The sequence has a different reading frame between nucleotides 1012 and 1035 (referenced to the previously published WIP sequence) due to CC pairs found at 1013 and 1036; the previously published sequence has a single C at 1013 and CCC at 1035. This altered reading frame results in the protein sequence 301-SASSQAPPPPPP-312 (the seven amino acids different from the previously published sequence are underlined). The sequence APPPPP is an ABM-2 motif. To verify this difference, a full-length WIP PCR product was obtained from a B cell cDNA library using primers flanking the WIP coding sequence. This product was cloned and was found to have the same sequence in this region as clone 9-1. In addition, RT-PCR of WIP nucleotides 810-1140 was performed using cDNA from mRNA obtained from PBMC from three different normal volunteer donors. Three different amplifications were performed from each RT reaction to exclude mutations introduced by PCR. PCR products were sequenced directly and showed the same sequence as our original clone 9-1. Taken together, these results show that the WIP sequence contains three ABM-2 motifs. In addition, a GGT to GCT change in codon 495 (nucleotide 1591 in AF031588) in the clone 9-1 results in a conservative Gly to Ala substitution. This substitution was also seen in the full-length PCR product obtained from the B cell library. This may represent an allelic difference.

View larger version (32K): [in this window] [in a new window]   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 µl of yeast suspensions (1 colony in 1 ml water) were spotted onto minimal media 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.

A number of missense mutations of WASP that cause WAS 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-hybrid system. Point mutations causing the amino acid substitutions R86H, Y107C, and A134T were introduced into the LexA-WASP 1–201 bait vector. These mutant bait strains, along with the empty bait vector negative control and the wild-type bait vector were transformed with empty library vector or our WIP clone 9-1. Fig. 2 shows the growth of these various strains on selective media. Impairment of growth of all three mutants on leucine-free media was observed, with the A134T mutant showing the most impairment. To further characterize the interaction, a liquid culture ß-galactosidase assay was performed as shown in Fig. 3. The assay showed impairment in ß-galactosidase production of all three WASP mutants after 3 h in induction media, and of A134T and R86H after 5 h. These results are consistent with the hypothesis that these mutations impair the interaction of WASP with WIP. The order of severity impairment is A134T > R86H > Y107C.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. WASP missense mutations interfere with WIP interaction as detected by growth on leu- media. Suspensions of yeast strains expressing either empty bait vector or LexA-WASP bait proteins with missense mutations (listed at the top) and either empty library vector or WIP clone 9-1 (listed to the left) were diluted to the OD 600 nm listed to the right of the plate photographs. A total of 10 µl 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 WASP with WIP.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. WASP missense mutations interfere with WIP interaction as detected by ß-galactosidase production. Yeast clones expressing the wild-type or mutant WASP bait proteins and WIP clone 9-1 were subjected to liquid culture ß-galactosidase assay as described in Materials and Methods. Enzyme activity detected at 3 and 5 h is indicated as absorbance at 420 nm, normalized to a culture OD 600 nm of 1.0. Three independent assays of each culture were performed and the results shown as the mean (bar) and the SD (whiskers). Impairment of ß-galactosidase production with the WASP missense mutations is evidence of impaired interaction of WASP with WIP. The results are representative of two experiments using different clones picked from the transformation plates.

View larger version (47K): [in this window] [in a new window]   FIGURE 4. In vitro assay of WASP-WIP interaction. Radiolabeled full-length WASP of wild-type or with the indicated missense mutations was incubated with GST alone or GST-WIP 416–503 coupled to glutathione Sepharose, and the bound proteins were analyzed by SDS-PAGE and autoradiography.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We found that the N-terminal 151 amino acids of WASP are important for WIP interaction. Since a number of mutations that cause WAS occur in this portion of the molecule, we tested several of these mutations to see if they impaired the WASP-WIP interaction. The mutation A134T strongly impaired WIP binding, and the Y107C mutation impaired it noticeably. Since the R86H mutation also impairs WIP binding, it seems that the configuration necessary for the interaction has contributions from throughout the N terminus of WASP. No data have yet been published regarding the secondary or tertiary structure of this region of WASP, and the effects of these mutations on the structure cannot be predicted. In terms of function, this region has been shown in N-WASP to bind to the phospholipid PIP2 (19). Based on homology with VASP and the mouse homologue of Drosophila enabled (Mena), this region may also bind a ligand related to the ABM-1 motif found in the Listeria ActA protein and in the cytoskeletal proteins vinculin and zyxin (21, 22, 23). The sequence of WIP from 416 to 503 that binds to WASP has several proline-rich motifs similar to the ABM-1 sequence that may mediate the WASP-WIP interaction. Perhaps the phospholipid 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 thrombocytopenia is still controversial. In general, missense mutations tend to cause mild disease characterized by thrombocytopenia and perhaps mild eczema, and null mutations tend to cause 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 exon 4 (encoding amino acids 121–154) tend to produce more severe disease. They reported that a patient with the mutation A134V, in the same codon as the mutation we tested here, had severe disease, yet made WASP in an EBV-transformed cell line at 60% of the normal level. This suggests that the mutation exerts a deleterious effect directly, by interfering with the WASP-WIP interaction, rather than indirectly, by affecting WASP protein stability. The A134T mutation tested here was also reported to have caused classical (moderate to severe) disease (14, 43); however, no information on the level of protein expression is available. Mutations in codon 86 are the most common missense mutations seen in WAS and can produce severe or mild disease (43, 47). A mutant WASP R86C was shown to be produced in detectable amounts in an EBV-transformed cell line, suggesting that it may also produce its effects directly (47). The Y107C mutation was reported to produce classical WAS (43) or attenuated disease (29); a patient with mild disease was observed to make low but detectable levels of the mutant protein in an EBV cell line (29). The argument that these missense mutations act directly is weakened somewhat by the findings of MacCarthy-Morrogh et al. (48). They show that in freshly isolated mononuclear cells from WAS patients with severe disease, protein was not detectable by immunoblot regardless of the type of mutation (missense vs null). Furthermore, they show that the amount of protein in an EBV-transformed cell line may not accurately reflect the amount of protein in the circulating cells of a patient. In general, patients with severe disease had no detectable circulating WASP, and patients with mild disease had detectable WASP.

Studies of actin-based motility in the pathogens Listeria and Shigella have been very fruitful in identifying cellular components involved in the production of actin filaments. The actin-related protein 2/3 (Arp2/3) complex, along with VASP, are bound by the Listeria protein ActA, which causes production of an actin tail that propels the bacterium through the cytoplasm (22, 49). Arp2/3 acts to produce actin filament nucleation, and VASP, by binding profilin, promotes actin polymerization. Neither Arp2/3 nor VASP binding alone is sufficient to produce bacterial motility. Recently, N-WASP has been shown to be required for actin-based motility of intracellular Shigella (50). The Shigella bacterial protein VirG binds N-WASP directly and VASP indirectly through the actin cross-linking protein vinculin. N-WASP may provide an actin nucleation activity, analogous to the activity of the Arp2/3 complex required for Listeria motility, through its ability to sever actin filaments. This activity, combined with the actin polymerization activity provided by VASP-bound profilin (and possibly by N-WASP or WIP-bound profilin) may fulfill the requirements for actin tail formation. WASP and N-WASP may be seen as proteins containing motifs providing both actin nucleation activity (verprolin homology, cofilin homology, and acidic residues region) and actin polymerization activity (intrinsic and WIP-associated profilin binding sites). These activities may then be localized, via interactions with PIP2 and/or SH3 domain proteins, to cellular sites targeted for assembly of actin filaments. The activities may also be regulated by interaction with SH3 domain proteins, CDC42, phosphorylation, and other yet to be described interactions.

In conclusion, we have shown for the first time that missense mutations that cause WAS impair the interaction of the disease-gene protein WASP with another protein. This protein, WIP, has motifs suggesting that it may act as a profilin-binding partner to WASP. The WASP-WIP interaction depends on residues throughout the N terminus of WASP. The need to clarify the details of the function of this complex region of WASP provides a direction for future research.

	Acknowledgments

 
We thank Drs. Colin Duckett, Luigi Notarangelo, and Fabio Candotti for their helpful review of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. David L. Nelson, Immunophysiology Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 4N-115, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address:

² Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; PIP2, phosphatidylinositol-4,5-biphosphate; EVH1, Ena/VASP homology 1; ABM, actin-based motility; SH3, Src homology 3; N-WASP, neural WASP; VH, verprolin homology; CH, cofilin homology; WIP, WASP interacting protein; GST, glutathione S-methyl transferase.

Received for publication November 23, 1998. Accepted for publication January 28, 1999.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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