WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.

Peptide recognition modules mediate many protein-protein interactions critical for the assembly of macromolecular complexes. Complete genome sequences have revealed thousands of these domains, requiring improved methods for identifying their physiologically relevant binding partners. We have developed a strategy combining computational prediction of interactions from phage-display ligand consensus sequences with large-scale two-hybrid physical interaction tests. Application to yeast SH3 domains generated a phage-display network containing 394 interactions among 206 proteins and a two-hybrid network containing 233 interactions among 145 proteins. Graph theoretic analysis identified 59 highly likely interactions common to both networks. Las17 (Bee1), a member of the Wiskott-Aldrich Syndrome protein (WASP) family of actin-assembly proteins, showed multiple SH3 interactions, many of which were confirmed in vivo by coimmunoprecipitation.

Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide ؉XXXPXXPX؉PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-Å resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.The actin cytoskeleton plays a key role in many essential cellular processes, such as motility, endocytosis, secretion, and membrane recycling (1-3). As a consequence, its organization and dynamic rearrangements need to be tightly controlled spatially and temporally. A thorough understanding of the interaction network connecting all the actin-associated proteins, the scaffolds and the anchoring proteins, is likely to help to clarify the mechanisms underlying its coordinated regulation (4).Most of the components of the yeast cell cytoskeleton have homologues in mammals where they often play similar roles (5-7). Abp1p 1 (actin-binding protein) is a Saccharomyces cerevisiae protein homologue to the mouse mAbp1p-SH3P7, which is a Src kinase target involved in polarized cell growth and motility (8, 9). The yeast Abp1 protein is 592 amino acids long and includes an actin depolymerizing factor-homology domain at the N terminus and a SH3 domain at the C terminus of the protein (10). Abp1p is found concentrated in the actin patches that are enriched at the sites of polarized cell surface growth in the bud of budding yeast and in the mating projection of mating yeast. The overexpression of Abp1p disturbs the actin cytoskeleton and leads to an aberrant budding pattern and cortical actin assembly (11). Deletion of the ABP1 gene, on the other hand, does not cause any apparent cytoskeletal defect (12). Abp1p, however, is found to be essential when any of the genes encoding for Sac6p (the actin filament-bundling protein, fimbrin), Sla1p, Sla2p, or Prk1p is deleted (13, 51). These proteins, which functionally interact with Abp1p, are also localized in the cortical actin cytoskeleton (11, 13). The observed negative synergism in yeast cells carrying combinations of deletions in ABP1-SH3 and in the SLA1, SLA2, and SAC6 genes suggests that these gene products perform redundant essential function(s) that require the presence of a...

The TOCA family of F-BAR–containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell–cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP–dependent actin-dynamics.

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Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.Key words: Binding speci¢city ; Molecular repertoire ; Phage display; Protein interaction module; SH3 domain Strategies to assemble a protein interaction mapGenomic databases, once translated into protein sequences, can be viewed as puzzle boxes waiting to be assembled into a coherent picture that resembles a cell. Given any protein segment, there are two types of strategies that are helpful in placing the corresponding protein in the appropriate slot in the cell puzzle (Fig. 1).The ¢rst approach is straightforward and comparable to the strategy utilized by 3-year-old children when they tackle their ¢rst jigsaw puzzle. It consists of a systematic screening of the limited repertoire of natural peptides in a search for partners that display a complementary surface. This approach does not involve any a priori understanding of the rules governing protein interaction. In principle, this would be the approach of choice but it is limited by the di¤culty of obtaining a complete, equally represented, collection of the proteins in a cell. It has been suggested that a high throughput implementation of the yeast two-hybrid method and of MALDI mass spectrometry could be exploited to develop a complete protein interaction map of an organism [1^4].On the other hand, one could take the alternative approach of deriving a set of rules that eventually would allow one to infer the binding partners from the primary structure of a related query protein. This second, perhaps more general, strategy could be implemented by an experimental approach that permits exploration of all the sequence space in order to extract the subset of sequences that have the potential to bind to the selected bait with an a¤nity above a certain threshold. In the hypothesis of a single solution, or a small subset of related solutions, to the problem of binding a given protein tar...