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At least 37 records · Page 2Linked to original sources
PubMed2001

Cytoprotection against mechanical forces delivered through beta 1 integrins requires induction of filamin A.

Cells in mechanically active environments can activate cytoprotective mechanisms to maintain membrane integrity in the face of potentially lethal applied forces. Cytoprotection may be mediated by expression of membrane-associated cytoskeletal proteins including filamin A, an actin-binding protein that increases the rigidity of the subcortical actin cytoskeleton. In this study, we tested the hypotheses that applied forces induce the expression of filamin A specifically and that this putative protective response inhibits cell death. Magnetically generated forces were applied to protein-coated magnetite beads bound to human gingival fibroblasts, cells with constitutively low basal levels of filamin A mRNA and protein. Forces applied through collagen or fibronectin, but not bovine serum albumin or poly-l-lysine-coated beads, increased mRNA and protein content of filamin A by 3-7-fold. Forces had no effect on the expression of other filamin isotypes or other cytoskeletal proteins. This effect was dependent on the duration of force and was blocked by anti-beta(1) integrin antibodies. Force also stimulated a 60% increase in expression of luciferase under the control of a filamin A promoter in transiently transfected Rat2 fibroblasts and was dependent on Sp1 transcription factor binding sites located immediately upstream of the transcription start site. Experiments with actinomycin D-treated cells showed that the increased filamin A expression after force application was due in part to prolongation of mRNA half-life. Antisense filamin oligonucleotides blocked force-induced filamin A expression and increased cell death by >2-fold above controls. The force-induced regulation of filamin A was dependent on intact actin filaments. We conclude that cells from mechanically active environments can couple diverse signals from forces applied through beta-integrins to up-regulate the production of cytoprotective cytoskeletal proteins, typified by filamin A.

Animals↗
PubMed2005

High affinity interaction with filamin A protects against calcium-sensing receptor degradation.

Calcium-sensing receptors (CaR) regulate cell proliferation, differentiation, and apoptosis through the MAPK pathway. MAPK pathway activation requires the cytoskeletal scaffold protein filamin A. Here we examine the interactions of CaR with filamin A in HEK-293 and M2 or A7 melanoma cells to determine how interactions with filamin A facilitate signaling. Filamin A interacts with CaR through two predicted beta-strands from residues 962 to 981; interactions between filamin A and CaR are greatly enhanced by exposure to 5 mM Ca2+. Truncations or deletions (from 972 to 997 or 962 to 981) of the CaR carboxyl terminus eliminate high affinity interactions with filamin A, but CaR-mediated MAPK pathway activation still occurs. CaR-mediated ERK phosphorylation can be localized to a predicted alpha-helix proximal to the membrane, which has been shown to be important for G protein-mediated signaling (residues 868-879). In M2 cells (-filamin A), CaR expression levels are very low; cotransfection of CaR with filamin A increases total cellular CaR and increases plasma membrane localization of CaR, facilitating CaR signaling to the MAPK pathway; similar results were obtained in HEK-293 cells. Interaction with filamin A increases cellular CaR by preventing CaR degradation, thereby facilitating CaR signaling. In addition, filamin A facilitates signaling to the MAPK pathway even by CaR truncations or deletion mutants that cannot engage in high affinity interactions with filamin A, suggesting the targeting of critical signaling proteins to CaR.

Cell Line↗
PubMed1999

Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

Actins↗
PubMed1983

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers.

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.

Actinin↗
PubMed2002

Dominant negative mutants of filamin A block cell surface expression of the D2 dopamine receptor.

Protein interaction screens have revealed an interaction between the D2 dopamine receptor and the actin cross-linking protein filamin A. However, the physiological significance of this interaction has not been explained. To better understand the role of filamin A in D2 receptor-mediated signaling, we examined the effect of disrupting filamin A/D2 receptor interaction. Overexpression of a truncated form of filamin A (repeat units 18-19 containing the D2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface D2 receptors. These results suggest that disruption of the linkage between D2 receptors and the actin cytoskeleton destabilizes plasma membrane-associated D2 receptors. Several missense mutations within repeat unit 19 of filamin A were identified that abrogate filamin A/D2 receptor interaction. Introduction of mutant and wild-type filamin A into filamin A-deficient M2 cells demonstrated that wild-type filamin A, but not the filamin A-binding mutants, was able to promote cell-surface expression of D2 receptors. Together, these studies provide evidence that filamin A/D2 receptor interaction is required for the proper targeting or stabilization of D2 dopamine receptors at the plasma membrane.

Amino Acid Sequence↗
PubMed2003

Filamin A binding to the cytoplasmic tail of glycoprotein Ibalpha regulates von Willebrand factor-induced platelet activation.

We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibalpha (GpIbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to GpIbalpha in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbalpha's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-GpIbalpha cytoplasmic domain fusion protein and competed with GpIbalpha for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbalpha regulates proaggregatory tyrosine kinase signaling.

Amino Acid Sequence↗
PubMed2005

The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins.

Myotilin and the calsarcin family member FATZ-1 (also called calsarcin-2 or myozenin-1) are recently discovered sarcomeric proteins implicated in the assembly and stabilization of the Z-discs in skeletal muscle. The essential role of myotilin in skeletal muscle is attested by the observation that certain forms of myofibrillar myopathy and limb girdle muscular dystrophy are caused by mutations in the human myotilin gene. Here we show by transfection, biochemical and/or yeast two-hybrid assay that: (1) myotilin is able to interact with the C-terminal region of FATZ-1 and that the N- or C-terminal truncations of myotilin abrogate binding; (2) myotilin can also interact with another calsarcin member, FATZ-2 (calsarcin-1, myozenin-2); (3) myotilin and FATZ-1 bind not only to the C-terminal region of filamin-C containing the Ig repeats 19-24, but also to the other two filamins, filamin-A and filamin-B, as well as the newly identified filamin-Bvar-1variant; (4) the binding of myotilin to filamin-C involves binding sites in its N-terminal region, whereas FATZ-1 associates with filamin-C via sequences within either its N- or C-terminal region; and finally, (5) the C-terminal region of filamin-C like filamin-B and filamin-Bvar-1, shows binding activity with the beta1A integrin subunit. Our findings further dissect the molecular interactions within the Z-disc that are essential for its organization, and provide evidence for a novel connection between Z-disc proteins and the sarcolemma via filamins and beta1 integrins. These data shed new light on the complex organization of the Z-disc that is highly relevant to understanding muscular dystrophies.

Animals↗
PubMed2000

Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin-binding protein filamin.

Kv4.2 potassium channels play a critical role in postsynaptic excitability. Immunocytochemical studies reveal a somatodendritic Kv4.2 expression pattern, with the channels concentrated mainly at dendritic spines. The molecular mechanism that underlies the localization of Kv4.2 to this subcellular region is unknown. We used the yeast two-hybrid system to identify the Kv4.2-associated proteins that are involved in channel localization. Here we demonstrate a direct interaction between Kv4.2 and the actin-binding protein, filamin. We show that Kv4.2 and filamin can be coimmunoprecipitated both in vitro and in brain and that Kv4.2 and filamin share an overlapping expression pattern in the cerebellum and cultured hippocampal neurons. To examine the functional consequences of this interaction, we expressed Kv4.2 in filamin(+) and filamin(-) cells and performed immunocytochemical and electrophysiological analyses. Our results indicate that Kv4.2 colocalizes with filamin at filopodial roots in filamin(+) cells but shows a nonspecific expression pattern in filamin(-) cells, with no localization to filopodial roots. Furthermore, the magnitude of whole-cell Kv4.2 current density is approximately 2.7-fold larger in filamin(+) cells as compared with these currents in filamin(-) cells. We propose that filamin may function as a scaffold protein in the postsynaptic density, mediating a direct link between Kv4.2 and the actin cytoskeleton, and that this interaction is essential for the generation of appropriate Kv4.2 current densities.

Actins↗
PubMed1997

H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway.

Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) for 1-60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H2O2. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H2O2 treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H2O2-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway.

Adenylyl Cyclases↗
PubMed1995

Actin filament cross-linking by chicken gizzard filamin is regulated by phosphorylation in vitro.

Filamin is a dimeric muscle phosphoprotein that cross-links actin filaments. We have found that purified chicken gizzard filamin is phosphorylated in vitro at serine residues by the Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Up to 0.9 mol of phosphate can be incorporated into 1 mol of filamin dimer. Phosphorylation by CaM kinase II increases filamin's critical actin filament gelling concentration and diminishes the amount of actin sedimented by filamin at low G-force. The modulation of filamin function by CaM kinase II requires ATP, Ca2+, and calmodulin, and it is abolished when CaM kinase II is inactivated with heat. Protein phosphatase 2A removed the phosphate added by CaM kinase II and restored filamin's actin filament cross-linking activity to the untreated basal level. In cosedimentation experiments, phosphorylation reduces the binding of filamin to actin filaments. The Kd for binding of filamin to actin filaments increases approximately 2-fold, from 3.2 to 6.9 microM, following CaM kinase II-mediated phosphorylation. Phosphorylation by CaM kinase II, therefore, regulates the binding of filamin to actin filaments.

Actins↗
PubMed1998

Human beta-filamin is a new protein that interacts with the cytoplasmic tail of glycoprotein Ibalpha.

We have cloned and sequenced a 9.4-kilobase cDNA specifying a new 280-kDa protein interacting with the cytoplasmic tail of glycoprotein (Gp) Ibalpha and showing considerable homology to actin-binding protein 280 (ABP-280) and chicken retinal filamin. We term this protein human beta-filamin. The gene for beta-filamin localizes to chromosome 3p14.3-p21.1. beta-Filamin mRNA expression was observed in many tissues and in cultured human umbilical vein endothelial cells (HUVECs); only minimal expression was detected in platelets and the megakaryocytic cell line CHRF-288. Like ABP-280, beta-filamin contains an NH2-terminal actin-binding domain, a backbone of 24 tandem repeats, and two "hinge" regions. A polyclonal antibody to the unique beta-filamin first hinge sequence identifies a strong 280-kDa band in HUVECs but only a weak band in platelets, and stains normal human endothelial cells in culture and in situ. We have confirmed the interaction of beta-filamin and GpIbalpha in platelet and HUVEC lysates. In addition, using two-hybrid analysis with deletion mutants, we have localized the binding domain for GpIbalpha in beta-filamin to residues 1862-2148, an area homologous to the GpIbalpha binding domain in ABP-280. beta-Filamin is a new member of the filamin family that may have significance for GpIbalpha function in endothelial cells and platelets.

Amino Acid Sequence↗
PubMed2003

Recovery from DNA damage-induced G2 arrest requires actin-binding protein filamin-A/actin-binding protein 280.

Filamin-A (filamin-1) is an actin-binding protein involved in the organization of actin networks. Our previous study shows that filamin-A interacts with BRCA2, and lack of filamin-A expression results in increased cellular sensitivity to several DNA damaging agents in melanoma cells (Yuan, Y., and Shen, Z. (2001) J. Biol. Chem. 276, 48318-48324), suggesting a role of filamin-A in DNA damage response. In this report, we demonstrated that deficiency of filamin-A results in an 8-h delay in the recovery from G2 arrest in response to ionizing radiation. However, filamin-A deficiency does not affect the initial activation of the G2/M checkpoint. We also found that filamin-A deficiency results in sustained activation of Chk1 and Chk2 after irradiation. This in turn causes a delay in the dephosphorylation of phospho-Cdc2, which is inhibitory to the G2/M transition. In addition, filamin-A-deficient M2 cells undergo mitotic catastrophe-related nuclear fragmentation after they are released from the G2 arrest. Together, these data suggest a functional role of filamin-A in the recovery from G2 arrest and subsequent mitotic cell death after DNA damage.

BRCA2 Protein↗
PubMed2003

SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V: localization of SHIP-2 to the activated platelet actin cytoskeleton.

The platelet receptor for the von Willebrand factor (VWF) glycoprotein Ib-IX-V (GPIb-IX-V) complex mediates platelet adhesion at sites of vascular injury. The cytoplasmic tail of the GPIbalpha subunit interacts with the actin-binding protein, filamin, anchoring the receptor in the cytoskeleton. In motile cells, the second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3) induces submembraneous actin remodeling. The inositol polyphosphate 5-phosphatase, Src homology 2 domain-containing inositol polyphosphate 5-phosphatase-2 (SHIP-2), hydrolyzes PtdIns(3,4,5)P3 forming phosphatidylinositol 3,4 bisphosphate (PtdIns(3,4)P2) and regulates membrane ruffling via complex formation with filamin. In this study we investigate the intracellular location and association of SHIP-2 with filamin, actin, and the GPIb-IX-V complex in platelets. Immunoprecipitation of SHIP-2 from the Triton-soluble fraction of unstimulated platelets demonstrated association between SHIP-2, filamin, actin, and GPIb-IX-V. SHIP-2 associated with filamin or GPIb-IX-V was active and demonstrated PtdIns(3,4,5)P3 5-phosphatase activity. Following thrombin or VWF-induced platelet activation, detection of the SHIP-2, filamin, and receptor complex decreased in the Triton-soluble fraction, although in control studies the level of SHIP-2, filamin, or GPIb-IX-V immunoprecipitated by their respective antibodies did not change following platelet activation. In activated platelets spreading on a VWF matrix, SHIP-2 localized intensely with actin at the central actin ring and colocalized with actin and filamin at filopodia and lamellipodia. In spread platelets, GPIb-IX-V localized to the center of the platelet and showed little colocalization with filamin at the plasma membrane. These studies demonstrate a functionally active complex between SHIP-2, filamin, actin, and GPIb-IX-V that may orchestrate the localized hydrolysis of PtdIns(3,4,5)P3 and thereby regulate cortical and submembraneous actin.

Actins↗
PubMed2005

Calpain 1-gamma filamin interaction in muscle cells: a possible in situ regulation by PKC-alpha.

Calpains are a family of calcium-dependent cysteine-proteases involved in cytoskeleton remodelling and muscle differentiation. In a recent study, we observed the presence of calpain 1 in the muscle contractile apparatus and specifically in the N1- and N2-lines. This calpain isoform was found to be involved in the degradation of muscle fibres via proteolysis of key proteins in Z-disk and costameric junctions. The goal of this study was to determine whether gamma-filamin--a specific muscle isoform of the filamin family--is a calpain 1 substrate and to characterise this interaction. Gamma-filamin is a major muscle architectural protein located in the Z-line and under the sarcolemmal membrane. This protein is a component of the chain binding the sarcolemma to the sarcomeric structure. In this study, we found that gamma-filamin formed a stable complex in vitro and in cells with calpain 1 in the absence of calcium stimulation. We also located the binding domains in the C-terminus of gamma-filamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2 region. The catalytic (80 kDa) and regulatory (28 kDa) subunits of calpain 1 are both involved in high affinity binding at gamma-filamin. Moreover, we showed that phosphorylation of the filamin C-terminus domain by PKC alpha protected gamma-filamin against proteolysis by calpain 1 in COS cells. Stimulation of PKC activity in myotubes, prevented gamma-filamin proteolysis by calpain and resulted in an increase in myotube adhesion.

Animals↗
PubMed1990

Purification of human smooth muscle filamin and characterization of structural domains and functional sites.

A method was developed to purify human smooth muscle filamin in high yield and structural domains were defined by using mild proteolysis to dissect the molecule into intermediate-sized peptides. Unique domains were defined and aligned by using high-resolution peptide mapping of iodinated peptides on cellulose plates. The amino- and carboxyl-terminal orientation of these domains within the molecule was determined by amino acid sequence analysis of several aligned peptides. In addition to the three unique domains which were identified, a number of smaller and larger fragments were also characterized and aligned within the intact molecule. These structural domains and related peptides provide a useful set of defined fragments for further elucidation of structure-function relationships. The two known functionally important binding sites of filamin, the self-association site and the actin-binding site, have been localized. Self-association of two monomers in a tail-to-tail orientation involves a small protease-sensitive region near the carboxyl terminal of the intact polypeptide chain. Sedimentation assays indicate that an actin-binding site is located near the blocked amino terminal of the filamin molecule. Sequences derived from large peptides mapping near the amino terminal show homology to the amino-terminal actin-binding site of alpha-actinin (chicken fibroblast and Dictyostelium), Dictyostelium 120-kDa actin gelation factor, beta-spectrin (human red cell and Drosophila), and human dystrophin. This homology is particularly interesting for two reasons. The functional form of filamin is single stranded, in contrast to alpha-actinin and spectrin which are antiparallel double-stranded actin cross-linkers. Also, no homology to the spectrin-like segments which comprise most of the mass of spectrin, alpha-actinin, and dystrophin was found. Instead, the sequence of a domain located near the center of the filamin molecule (tryptic 100-kDa peptide, T100) shows homology to the published internal repeats of the Dictyostelium 120-kDa actin gelation factor. On the basis of these results, a model of human smooth muscle filamin substructure is presented. Also, comparisons of human smooth muscle filamin, avian smooth muscle filamin, and human platelet filamin are reported.

Amino Acid Sequence↗
PubMed2000

Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis.

We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein.

In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis of the interactions of known signaling proteins. Recently, scaffolding and adaptor proteins have been identified that organize signaling proteins in cells and that contribute to the nature and specificity of signaling pathways. In an effort to identify proteins that might organize the signaling system(s) activated by the extracellular Ca(2+) receptor (CaR), we used a bait construct representing the intracellular C terminus of the human CaR and the yeast two hybrid system to screen a human kidney cDNA library. We identified a clone representing the C-terminal 1042 amino acids (aa) of the cytoskeletal protein filamin (ABP-280). Analysis of truncation and deletion constructs of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907-997 of the CaR C terminus and aa 1566-1875 of filamin. Interaction of the two proteins in mammalian HEK-293 cells was demonstrated by co-immunoprecipitation and colocalization of them using immunofluorescence microscopy. The functional importance of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably express filamin, and demonstrating that the CaR activated ERK only in the presence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the ability of the CaR to activate p42ERK in a dose-dependent manner, but did not inhibit the ability of the ET(A) receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen-activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR.

Amino Acid Sequence↗
PubMed2003

The F-actin cross-linking and focal adhesion protein filamin A is a ligand and in vivo substrate for protein kinase C alpha.

Filamin A is an established structural component of cell-matrix adhesion sites. In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules. Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesion-dependent signaling have remained elusive. We performed a yeast two-hybrid analysis using protein kinase Calpha as a bait and identified filamin as a direct binding partner. The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin. In vitro ligand binding assays revealed a Ca2+ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites. Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B. PKC-dependent phosphorylation of filamin was also detected in cells. Our data suggest an intimate interaction between filamin and PKC in cell signaling.

Actins↗