Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.     

Structural basis of the migfilin-filamin interaction and competition with integrin beta tails

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro(5) and Pro(19) and binds to the CD face of the IgFLNa21 beta-sandwich. This interaction is similar to the previously characterized beta 7 integrin-IgFLNa21 interaction and migfilin and integrin beta tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.     

Structure of three tandem filamin domains reveals auto-inhibition of ligand binding

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.     

The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended

Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.     

Cystic Fibrosis Transmembrane Conductance Regulator Interacts with Multiple Immunoglobulin Domains of Filamin A

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa·partner complexes as templates, we generated in silico models of IgFLNa·CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated (∼160 nm) strands, whereas CFTR is compact (6∼8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa·partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.     

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.     

1H, 13C and 15N resonance assignments of the human filamin A tandem immunoglobulin-like domains 16–17 and 18–19

Filamins are large actin-binding and cross-linking proteins which act as linkers between the cytoskeleton and various signaling proteins. Filamin A (FLNa) is the most abundant of the three filamin isoforms found in humans. FLNa contains an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains. The Ig domains are responsible for the FLNa dimerization and most of the interactions that FLNa has with numerous other proteins. There are several crystal and solution structures from isolated single Ig domains of filamins in the PDB database, but only few from longer constructs. Here, we present nearly complete chemical shift assignments of FLNa tandem Ig domains 16–17 and 18–19. Chemical shift mapping between FLNa tandem Ig domain 16–17 and isolated domain 17 suggests a novel domain–domain interaction mode.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.     

Filamins Regulate Cell Spreading and Initiation of Cell Migration

Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding member of the family, was implicated in migration by cell biological analyses and the identification of FLNA mutations in the neuronal migration disorder periventricular heterotopia. However, recent knockout studies have questioned the relevance of FLNa to cell migration. Here we have used shRNA-mediated knockdown of FLNa, FLNb or FLNa and FLNb, or, alternatively, acute proteasomal degradation of all three FLNs, to generate FLN-deficient cells and assess their ability to migrate. We report that loss of FLNa or FLNb has little effect on migration but that knockdown of FLNa and FLNb, or proteolysis of all three FLNs, impairs migration. The observed defect is primarily a deficiency in initiation of motility rather than a problem with maintenance of locomotion speed. FLN-deficient cells are also impaired in spreading. Re-expression of full length FLNa, but not re-expression of a mutated FLNa lacking immunoglobulin domains 19 to 21, reverts both the spreading and the inhibition of initiation of migration.Our results establish a role for FLNs in cell migration and spreading and suggest that compensation by other FLNs may mask phenotypes in single knockout or knockdown cells. We propose that interactions between FLNs and transmembrane or signalling proteins, mediated at least in part by immunoglobulin domains 19 to 21 are important for both cell spreading and initiation of migration.     

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K_a ~ 10⁵), the binding of the intact protein to F-actin is strong (apparent K_a ~ 10⁸), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat–F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.     

Identification and Characterization of Multiple Similar Ligand-binding Repeats in Filamin: IMPLICATION ON FILAMIN-MEDIATED RECEPTOR CLUSTERING AND CROSS-TALK*

The actin-binding protein filamin links membrane receptors to the underlying cytoskeleton. The cytoplasmic domains of these membrane receptors have been shown to bind to various filamin immunoglobulin repeats. Notably, among 24 human filamin repeats, repeat 17 was reported to specifically bind to platelet receptor glycoprotein Ibα and repeat 21 to integrins. However, a complete sequence alignment of all 24 human filamin repeats reveals that repeats 17 and 21 actually belong to a distinct filamin repeat subgroup (containing repeats 4, 9, 12, 17, 19, 21, and 23) that shares a conserved ligand-binding site. Using isothermal calorimetry and NMR analyses, we show that all repeats in this subgroup can actually bind glycoprotein Ibα, integrins, and a cytoskeleton regulator migfilin in similar manners. These data provide a new view on the ligand specificity of the filamin repeats. They also suggest a multiple ligand binding mechanism where similar repeats within a filamin monomer may promote receptor clustering or receptor cross-talking for regulation of the cytoskeleton organization and diverse filamin-mediated cellular activities.     

Homology modeling and consensus protein disorder prediction of human filamin

Filamins are dimeric actin-binding proteins participating in the organization of the actin-based cytoskeleton. Their modular domain organization is made up of an N-terminal actin-binding domain composed of two CH domains followed by flexible rod regions that consist of 24 Ig-like domains. Homology modeling was used to model human filamin using Modeller 9v5. The resulting model assessed by Verify 3D and PROCHECK showed that the final model is reliable. The conformational disorder prediction of human filamin residues were also mapped on the validated structure of human filamin. Prediction of protein disorder in filamin structures will help structural biologists to find suitable targets to be analyzed and for understanding protein function.     

Epidermal Growth Factor (EGF) Regulates α5β1 Integrin Activation State in Human Cancer Cell Lines through the p90RSK-dependent Phosphorylation of Filamin A

Cell adhesion, motility, and invasion are regulated by the ligand-binding activity of integrin receptors, transmembrane proteins that bind to the extracellular matrix. Integrins whose conformation allows for ligand binding and appropriate functional activity are said to be in an active state. Integrin activation and subsequent ligand binding are dynamically regulated by the association of cytoplasmic proteins with integrin intracellular domains. In this study, we evaluated the role of EGF in the regulation of the activation state of the α5β1 integrin receptor for fibronectin. The addition of EGF to either A431 squamous carcinoma cells or DiFi colon cancer cells resulted in loss of α5β1-dependent adhesion to fibronectin but no loss of integrin from the cell surface. EGF activated the EGF receptor/ERK/p90RSK and Rho/Rho kinase signaling pathways. Blocking either pathway inhibited EGF-mediated loss of adhesion, suggesting that they work in parallel to regulate integrin function. EGF treatment also resulted in phosphorylation of filamin A (FLNa), which binds and inactivates β1 integrins. EGF-mediated FLNa phosphorylation was completely blocked by an inhibitor of p90RSK and partially attenuated by an inhibitor of Rho kinase, suggesting that both pathways converge on FLNa to regulate integrin function. A431 clonal cell lines expressing non-phosphorylated dominant-negative FLNa were resistant to the inhibitory effects of EGF on integrin function, whereas clonal cell lines overexpressing wild-type FLNa were more sensitive to the inhibitory effect of EGF. These data suggest that EGF-dependent inactivation of α5β1 integrin is regulated through FLNa phosphorylation and cellular contractility.     

Adapter protein SH2B1beta binds filamin A to regulate prolactin-dependent cytoskeletal reorganization and cell motility

Prolactin (PRL) regulates cytoskeletal rearrangement and cell motility. PRL-activated Janus tyrosine kinase 2 (JAK2) phosphorylates the p21-activated serine-threonine kinase (PAK)1 and the Src homology 2 (SH2) domain-containing adapter protein SH2B1β. SH2B1β is an actin-binding protein that cross-links actin filaments, whereas PAK1 regulates the actin cytoskeleton by different mechanisms, including direct phosphorylation of the actin-binding protein filamin A (FLNa). Here, we have used a FLNa-deficient human melanoma cell line (M2) and its derivative line (A7) that stably expresses FLNa to demonstrate that SH2B1β and FLNa are required for maximal PRL-dependent cell ruffling. We have found that in addition to two actin-binding domains, SH2B1β has a FLNa-binding domain (amino acids 200-260) that binds directly to repeats 17-23 of FLNa. The SH2B1β-FLNa interaction participates in PRL-dependent actin rearrangement. We also show that phosphorylation of the three tyrosines of PAK1 by JAK2, as well as the presence of FLNa, play a role in PRL-dependent cell ruffling. Finally, we show that the actin- and FLNa-binding-deficient mutant of SH2B1β (SH2B1β 3Δ) abolished PRL-dependent ruffling and PRL-dependent cell migration when expressed along with PAK1 Y3F (JAK2 tyrosyl-phosphorylation-deficient mutant). Together, these data provide insight into a novel mechanism of PRL-stimulated regulation of the actin cytoskeleton and cell motility via JAK2 signaling through FLNa, PAK1, and SH2B1β. We propose a model for PRL-dependent regulation of the actin cytoskeleton that integrates our findings with previous studies.     

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.     

Indications for a novel muscular dystrophy pathway. gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin

gamma-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although gamma-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of gamma-filamin. The two Ig-like domains of the recently described alpha-actinin-binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of gamma-filamin may be indirectly anchored to alpha-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.     

利用RNA干扰抑制细丝蛋白A基因的表达

目的：构建细丝蛋白A（FLNa）基因的小干扰RNA（siRNA）表达载体,并观察其对FLNa基因表达的抑制作用。方法：利用RNA干扰（RNAi）技术设计并合成1条针对FLNa的siRNA,将其克隆到siRNA表达载体pSilencer4．1-CMV-hygro中;将重组质粒pSilencer-FLNa、pSilencer-negative（阴性对照）转染293T人胚肾细胞,通过Western印迹检测FLNa的表达;通过潮霉素筛选建立干扰FLNa表达的前列腺癌细胞。结果：PCR鉴定证明构建了FLNa基因RNAi载体;Western印迹表明构建的FLNa基因干扰载体能够有效地抑制FLNa基因的表达;建立了稳定干扰FLNa表达的前列腺癌C4-2细胞。结论：构建了FLNa基因RNAi载体,该载体能够有效地抑制FLNa基因的表达。     

FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling

FilGAP is a newly recognized filamin A (FLNa)-binding RhoGTPase-activating protein. The GTPase-activating protein (GAP) activity of FilGAP is specific for Rac and FLNa binding targets FilGAP to sites of membrane protrusion, where it antagonizes Rac in vivo. Dominant-negative FilGAP constructs lacking GAP activity or knockdown of endogenous FilGAP by small interference RNA (siRNA) induce spontaneous lamellae formation and stimulate cell spreading on fibronectin. Knockdown of endogenous FilGAP abrogates ROCK-dependent suppression of lamellae. Conversely, forced expression of FilGAP induces numerous blebs around the cell periphery and a ROCK-specific inhibitor suppresses bleb formation. ROCK phosphorylates FilGAP, and this phosphorylation stimulates its RacGAP activity and is a requirement for FilGAP-mediated bleb formation. FilGAP is, therefore, a mediator of the well-established antagonism of Rac by RhoA that suppresses leading edge protrusion and promotes cell retraction to achieve cellular polarity.