Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5679–5684, May 1997 Cell Biology The IyLWEQ module: a conserved sequence that signifies F-actin binding in functionally diverse proteins from yeast to mammals RICHARD O. MCCANN*† AND SUSAN W. CRAIG*‡ Departments of *Biological Chemistry and ‡Pathology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205 Communicated by Thomas D. Pollard, Salk Institute for Biological Studies, La Jolla, Ca, March 27, 1996 (received for review January 21, 1997) ABSTRACT Talin is an actin-binding protein involved in functions associated with individual talin domains are dis- integrin-mediated cell adhesion and spreading. The C- persed among proteins having cellular roles distinct from those terminal 197 amino acids of vertebrate talin are 45% similar of talin. These proteins appear to fall into three groups. The to the C-terminal residues of Sla2, a yeast protein implicated first of these is the erythrocyte band 4.1 superfamily. Talin is in polarized assembly of the yeast actin cytoskeleton. Talin is a member of this group based on similarity between the 47-kDa also homologous in this region to nematode talin, cellular fragment of talin and the N-terminal region of erythrocyte slime mold filopodin, and an Sla2 homolog from nematode. band 4.1 (1). The 4.1 superfamily also includes the related Analysis of the conserved C-terminal sequences of these five cytoskeletal proteins ezrin, moesin, and radixin, the tumor proteins with BLOCK MAKER reveals a series of four blocks, suppressor schwannomin (NF-2), and several protein tyrosine which we name the IyLWEQ module after the conserved initial phosphatases (20). All of these proteins are related by a residues in each block. Experiments presented here show that homologous 4.1-like domain. All members of the 4.1 super- the conserved protein domain represented by the IyLWEQ family are known, or potential, membrane-associated proteins. module competes quantitatively with native talin for binding A second group of proteins contains members that are to F-actin in vitro. Furthermore, the corresponding domain of similar to vertebrate talin at their N-terminal 4.1 region and at Sla2 binds to both yeast and vertebrate F-actin in vitro. their C termini. One such talin homolog has been identified in Mutation of one of the conserved residues in the fourth Dictyostelium (21). This 2,491-residue protein, filopodin, ac- conserved block abolishes the interaction of the Sla2 IyLWEQ cumulates in response to chemoattractant at the leading edge module with F-actin. These results establish the location of an of motile cells, where F-actin is also enriched. Other than at F-actin binding domain in native talin, demonstrate that their N and C termini, talin and filopodin are not very similar. direct interaction of Sla2 with actin is a possible basis for its In contrast to filopodin, an apparently bona fide talin has been effect on the actin cytoskeleton in vivo, and define the I LWEQ y identified in Caenorhabditis elegans (22). This 2,553-residue consensus as a new actin-binding motif. talin is 59% similar to mouse talin over its entire length, with greatest similarity at its N terminus, including the band 4.1 Talin is a modular protein (1, 2) found in focal adhesions (3), region, and at its C terminus. multiprotein assemblies mediating interactions between the A third group of proteins is homologous to talin only at the actin cytoskeleton of cultured cells and the extracellular C-terminal 200 residues. This group is represented by Sla2, a environment (4). Perturbation of talin in vivo indicates that 968-residue yeast protein that is required for polarized assem- talin functions in cell adhesion and spreading (5–7). Experi- bly of the actin cytoskeleton (23). SLA2 is allelic with END4, ments with purified proteins demonstrate that talin interacts whose product is required for endocytosis (24), and with with several focal adhesion components, including acidic phos- MOP2, which is necessary for the proper plasma membrane pholipids (8), actin (9–11), vinculin (12), b1 integrin (13), b3 1 integrin (14), and focal adhesion kinase (FAK) (15). Talin has localization of the H -ATPase encoded by PMA1 (25). Talin a calculated monomer mass of 269 kDa and is a dimer in its and Sla2 (End4yMop2) also share their similar C terminus actin-binding form (16, 17) and at protein concentrations with a putative Sla2 protein from C. elegans (26). greater than 1 mM (16). Calpain cleaves talin into two frag- Because Sla2 is required for nucleated assembly of actin in ments that have different activities in vitro. The 190-kDa, a permeabilized yeast cell model (27), and because talin both C-terminal fragment binds to G and F-actin (2, 9) and to nucleates actin polymerization and binds to F-actin in vitro (9, vinculin (18), and nucleates actin polymerization (17). Recent 10, 17), we hypothesized that Sla2 might interact directly with studies with glutathione S-transferase (GST) fusion proteins actin and that the actin remodeling activities of talin and Sla2 provide evidence for two, nonoverlapping F-actin binding reside in their conserved C-terminal domain. To test this idea, sequences in the 190-kDa talin fragment (19), and one F-actin we constructed two GST-fusion proteins incorporating the binding sequence from the N-terminal, 47-kDa region. In C-terminal 197 residues of mouse talin and the homologous other studies, the 47-kDa proteolytic fragment did not bind to residues of yeast Sla2 and evaluated their potential to interact F-actin or nucleate actin polymerization (2, 17). The 47-kDa with vertebrate and yeast actin. fragment contains all of the acidic phospholipid binding activity of intact talin (2), which has led to the hypothesis that MATERIALS AND METHODS the N terminus of talin mediates talin-membrane interactions in vivo. Protein Preparation. Rabbit skeletal muscle actin was pu- Several proteins have been identified that are similar to one rified as described (28), with an additional gel filtration step or more limited regions of talin, suggesting that the specific (29). Pyrene-labeled actin was produced by labeling F-actin with pyrene iodoacetamide (30, 31). Yeast actin (32) and The publication costs of this article were defrayed in part by page charge chicken gizzard talin (33) were purified as described. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Sequence Analysis. The C-terminal sequences of accordance with 18 U.S.C. §1734 solely to indicate this fact. talin and its putative homologs were aligned and blocks 1–4 of Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y945679-6$2.00y0 Abbreviation: GST, glutathione S-transferase. PNAS is available online at http:yywww.pnas.org. †To whom reprint requests should be addressed. 5679 Downloaded by guest on September 25, 2021 5680 Cell Biology: McCann and Craig Proc. Natl. Acad. Sci. USA 94 (1997) the IyLWEQ module identified using BLOCK MAKER (34) Airfuge) for 20 min and the supernatant and pellet were [www.blocks.fhcrc.orgy]. separated for subsequent analysis on SDSyPAGE. GST Fusion Proteins. The mouse talin GST fusion protein F-Actin Bundling. Actin (3 mM) was polymerized in the construct (GST-Tn.2345–2541) was prepared by amplifying presence of GST, GST-Tn.2345–2541, or GST-Sla2.771–968 (3 the coding region extending from I2345 to the stop codon, using mM each). The resulting F-actin preparations were adsorbed to a mouse talin cDNA as the template, and subcloning this PCR grids, negatively stained with uranyl formate, and visualized on fragment into pCR 2.1 (Invitrogen). The following primers a Zeiss model 10A electron microscope. were used: 59-ATCCTAGAAGCTGCC-39 (7192–7206; Nucleation of Actin Polymerization. G-actin (6 mM, 54% pyrene-labeled) was polymerized as outlined above in the Start5I2345); 59-TTAGTGCTCGTCTCG-39 [7785–7771; numbering corresponds to that in Rees et al. (1)]. The EcoRI presence of talin, GST-Tn, GST-Sla2, or GST (2 mM each), insert-containing fragment from pCR2.1 was then subcloned except for the addition of 30 mM NaCl and a final Tris into EcoRI-digested pGEX-2T (Pharmacia). The Sla2 GST concentration of 7 mM. Polymerization was monitored by the fusion protein construct (GST-Sla2.771–968) was prepared increase in pyrene fluorescence (Perkin–Elmer model LS 50 B; similarly, except that yeast genomic DNA (strain SM1060) was excitation: 365 nm; emission 407 nm). Gelsolin (2 nM) was used as a positive control for nucleation. used as the template. The following primers were used for the Sla2 construct: 59-CCATTGTTGTCATTGGC-39 (Chr XIV: 190360–190376; Start5P771); 59-GATCAATCATCATC- RESULTS CTGG-39 (Chr XIV: 190958–190944; numbering from the At the start of this study, BLOCK MAKER (34) was used to aid Saccharomyces Genome Database). The fusion proteins were in identification of the most conserved patterns of amino acids purified using glutathione-agarose as previously described in the conserved C termini of the four proteins then identified (35). as talin homologs (1, 21, 23, 26). BLOCK MAKER uses automated F-Actin Cosedimentation. Cosedimentation of mouse talin, versions of MOTIFS (36) and GIBBS (37) in conjunction with GST-Tn.2345–2541, and GST-Sla2.772–968 with F-actin was MOTOMAT (38) for identification of the best set of conserved measured essentially as described in Schmidt et al. (11) by amino acid patterns (motifs) in homologous protein sequences. mixing G-actin (in buffer G: 2 mM Tris, pH 7.5, 9490.2 mM The MOTIFS algorithm with the parameters s, r, and d (36) CaCl2y0.2 mM ATPy0.2 mM dithiothreitol) with talin or one automatically fixed at 4, 0, and 17, respectively, found blocks of the GST fusion proteins and then initiating actin polymer- 1–4 (Fig.