The I/LWEQ module: a conserved sequence that signifies F-actin binding in functionally diverse proteins from yeast to mammals

McCann, Richard O.; Craig, Susan W.

doi:10.1073/pnas.94.11.5679

Cited by 175 publications

(190 citation statements)

References 47 publications

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“…These observations are consistent with experiments in i o showing that integrins are key determinants of the subcellular localization of talin [21,22]. Talin is also an F-actinbinding protein [13,23,24] and is reported to possess activity in both actin nucleation [25] and actin bundling [26]. Therefore talin has the potential to link integrins directly to the actin cytoskeleton.…”

Section: Introductionsupporting

confidence: 87%

“…Such vinculin-VASP complexes might be able to bind to sites in both talin subunits, cross-linking the talin dimer. Our previous results provided evidence that talin also has more than one actin-binding site [13], although the best characterized of these is that towards the Cterminus of the protein [9,13,24]. In an anti-parallel dimer the Cterminal actin-binding site in talin would be close to the vinculin-binding sites ( Figure 7B).…”

Section: Figure 7 Domain Structure Of Talinmentioning

confidence: 93%

“…(A) The domain structure of talin, showing the positions of the binding sites for pp125 focal adhesion kinase, layilin [3], vinculin [27][28][29] and F-actin [13,23,24]. (B) The possible relationship between the three vinculin-binding sites and the C-terminal actin-binding site in a talin homodimer.…”

Section: Figure 7 Domain Structure Of Talinmentioning

confidence: 99%

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Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

et al. 1999

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Using recombinant talin polypeptides and an SDS\PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin [Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J. Cell Biol. 122, 337-347]. We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969 ; alignment of these sequences shows 59 % similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic α-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two

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Section: Introductionsupporting

confidence: 87%

Section: Figure 7 Domain Structure Of Talinmentioning

confidence: 93%

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Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

et al. 1999

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“…Additionally, at pH 6.5 the K d of 2.92 Ϯ 0.62 M was significantly lower compared with a K d of 8.68 Ϯ 3.91 M at pH 7.5 (P Ͻ 0.01, n ϭ 3). Although the I/LWEQ domain is a conserved actinbinding module also found in the fungal protein Sla2p, Dictyostelium TalA and TalB, and metazoan proteins Hip1 (Huntington-interacting protein 1) and Hip12, (16), the adjacent USH segment was previously reported to inhibit F-actin binding in vitro by the I/LWEQ domain (17). However, attenuated actin binding by the USH segment was determined at pH 8, which is consistent with our data indicating that the USH segment plays a pivotal role in conferring decreased affinity for F-actin binding by the I/LWEQ domain at higher pH values.…”

Section: Decreased Ph Increases the Affinity Of F-actin Binding By Tamentioning

confidence: 99%

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Srivastava

Barreiro

Groscurth

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

133

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Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pHi) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pKa values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pHi decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin. intracellular pH ͉ NHE1 ͉ migration F ocal adhesion (FA) remodeling is a rate-limiting determinant in haptokinetic migration of adherent cells. At the leading edge of migrating cells, FAs undergo rapid cycles of assembly and turnover, creating and disrupting, respectively, sites of traction necessary for forward movement of the cell body. Force generation for traction requires linkage among the extracellular matrix, integrin receptors, and actin filaments. Actin filaments do not directly bind to the cytoplasmic domain of integrins but bind to integrin-associated FA proteins such as talin and vinculin. Although several mechanisms contribute to FA remodeling in migrating cells (1), emerging evidence indicates that talin plays a central role in the dynamic linkage between integrins and actin filaments necessary for cell migration (2, 3). Talin functions in distinct albeit complementary mechanisms that promote FA turnover. First is cleavage of talin by the protease calpain, which also modulates adhesion complex composition and likely signaling functions of talin (4). Second is regulated talin binding to actin filaments, which is proposed to act as a clutch to control FA turnover and membrane protrusion dynamics (3, 5-7). How actin binding by talin is dynamically regulated during cell migration, however, remains undetermined.Previous stu...

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“…Potential candidates for mediating the membrane-actin coupling are the yeast protein Sla2 and its mammalian homolog HIP1R because (i) they can bind both the membrane and actin via their N-terminal ANTH domain and C-terminal THATCH/talin-like domain, respectively (7)(8)(9)(10), and (ii) cells deficient in these proteins exhibit a specific endocytic block called an "uncoupling phenotype." The knockout of the SLA2 gene in yeast (11) or siRNAmediated down-regulation of HIP1R in HeLa cells (12) stops endocytic vesicle budding even though the endocytic coats are assembled on the plasma membrane and actin polymerizes continuously at these sites.…”

mentioning

confidence: 99%

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

Skružný

Brach

Ciuffa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

144

209

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Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane-actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2's ANTH and Ent1's ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a liganddependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.ANTH domain | membrane remodeling | PIP 2

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The I/LWEQ module: a conserved sequence that signifies F-actin binding in functionally diverse proteins from yeast to mammals

Cited by 175 publications

References 47 publications

Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

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