Filamin inhibits actin activation of heavy meromyosin ATPase.
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Actins↗
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FAP52 - a new partner for filamin in actin cytoskeleton organization.
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Actins↗
A comparison of methods used to characterize gelation of actin in vitro.
We have compared the meniscus depletion assay and falling ball viscometry, two means of assessing the extent of gelation in actin-based systems using mixtures of actin and the actin-binding protein filamin. We examined the effect of varying the concentrations of actin and filamin in both assays. The interaction of actin and filamin was detected only above a threshold concentration of filamin. This threshold concentration was lower for falling ball viscometry than for the meniscus depletion assay at equal actin concentrations. At constant concentrations of filamin, an increase in actin concentration caused an increase in apparent viscosity measured by the falling ball assay, but a decrease in sedimentability detected by the meniscus depletion assay. The rate of sedimentation of actin was dependent on the molar ratio of actin to filamin. At each molar ratio, the sedimentation of actin was not dependent on the specific concentrations of actin and filamin used. The apparent viscosity was dependent on both the molar ratio and the specific concentrations of actin and filamin. To relate the present results to earlier studies, we examined mixtures of actin and filamin using a macroscopic assay of gelation (tube tipping assay), and polarized light microscopy. The effect of increasing filamin concentration in the four assays was compared at three actin concentrations. Mixtures of actin and filamin whose apparent viscosities were low enough to be estimated by falling ball viscometry were optically isotropic fluids that flowed out of inverted test tubes. Mixtures of actin and filamin in the range of sensitivity of the meniscus depletion assay were either viscous fluids or gels, and were either optically isotropic or anisotropic. Thus, the four assays provide different estimates of gelation. Both the meniscus depletion assay and falling ball viscometry can be used to determine relative gelation activity, but neither can be used as a quantitative assay of gelation.
Actins↗
Regulation of tension-induced mechanotranscriptional signals by the microtubule network in fibroblasts.
Mechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through beta1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured fibroblasts at focal adhesions. Force enhanced recruitment of alpha-tubulin and the plus end microtubule-binding protein cytoplasmic linker protein-170 (CLIP-170) at focal adhesions. Immunoprecipitation studies demonstrated no direct binding of tubulin to actin or filamin A, but CLIP-170 interacted with tubulin, filamin A, and beta-actin. The association of CLIP-170 with beta-actin was enhanced by force. Force activated the p38 mitogen-activated protein kinase, increased filamin A expression, and induced the relocation of p38 and filamin A to focal adhesions. Disruption of microtubules with nocodazole, independent of force application, enhanced filamin A expression and Sp1-mediated filamin A promoter activity, while stabilization of microtubules with Taxol inhibited force induction of both filamin A mRNA and protein. We conclude that in response to tensile forces applied through beta1 integrins and actin the microtubule network modulates mechanotranscriptional coupling of filamin A.
Actins↗
Tumor cell locomotion and metastatic spread.
The cytoskeletal filament proteins alpha-actinin, filamin, desmin, and filamin-desmin aggregates were adsorbed to a hydrophobic silica surface. The adsorbed amount as measured by ellipsometric methods after rinsing and equilibration was 2.7 mg/m2 for alpha-actinin and 0.4 mg/m2 for filamin plus desmin, respectively. Adsorbed layer thicknesses in physiological salt solution were about 107 nm, 89 nm, 108 nm and 93 nm for alpha-actinin, filamin, desmin, and cross-linked filamin-desmin, respectively. Ca2+ ions in a concentration of 10(-4), 10(-3), and 2.52 mmol/l had no effect on the adsorbed amount, refractive index, and adsorbed layer thickness of the individual intermediate filament proteins. Cross-linked filamin-desmin, however, reacted markedly upon the addition of these Ca2+ concentrations with a change in refractive index and adsorbed layer thickness. The layer formed by the filamin-desmin complex contracted by 2-3, 6-7, and 6-7 nm, respectively. The maximum shortening occurred at 1 pmol/l Ca2+. The Ca(2+)-dependent adsorbed layer changes of cross-linked filamin-desmin supports the contractile mechanisms in muscular tissues and forms the basis for migration and motility in nonmuscular cells. These motional events are crucially involved in peripheral organ perfusion, inflammation, and tumor invasion and metastasis.
Adsorption↗
Emerin deletions occurring on both Xq28 inversion backgrounds.
Emery-Dreifuss muscular dystrophy (EMD) is an X-linked disorder characterized by contractures, progressive weakness and cardiomyopathy. EMD is caused by mutations in the 2 kb emerin gene that is located within human Xq28. Emerin is immediately distal to the 26 kb filamin gene, and flanking the filamin-emerin region are two large inverted repeats. This entire region previously has been found to be inverted in approximately 20% of X chromosomes, presumably mediated by the inverted repeats. Only one complete emerin deletion has been reported previously. It was found to be due to a complex rearrangement involving the inverted repeats which partially duplicated filamin. We report here two additional EMD patients who have large deletions of 20 and 34 kb, respectively. Unlike the previously reported deletion, these deletions appear to be simple deletions, with each breakpoint junction showing only 2 bp of overlap, suggesting an end-joining mechanism. However, the two deletions were found on each of the two inverted backgrounds. The 20 kb deletion includes the entire emerin gene and extends well into most of the distal inverted repeat. In contrast, the 34 kb deletion occurs on the inverted X chromosome and extends centromeric, well beyond the proximal inverted repeat. In addition, at least three nearby putative genes detected by previous sequence analysis are deleted among these patients but without obvious deviation from a typical EMD phenotype. Similarly to the previously reported deletion, filamin remains intact in these two deletions. All three deletions involve distinct breakpoints within the 4.7 kb filamin-emerin intergenic region, suggesting that loss of filamin is a lethal event. Thus, the close proximity of filamin to emerin may place constraints upon potential emerin deletions and probably accounts for the rarity of complete emerin deletions in EMD patients.
Chromosome Inversion↗
A novel anti-inflammatory peptide inhibits endothelial cell cytoskeletal rearrangement, nitric oxide synthase translocation, and paracellular permeability increases.
The endothelial cell (EC) membrane-cytoskeletal interface in part maintains plasma membrane integrity and promotes cell-cell apposition. Nonmuscle filamin (ABP-280), an actin crosslinking protein, promotes orthogonal branching of F-actin and is the major protein that links the peripheral actin network to the plasma membrane through its C-terminal glycoprotein binding site. In response to bradykinin, filamin translocates from the cell periphery to the cytosol within 1 min. A synthetic peptide, corresponding to filamin's C-terminal calcium/calmodulin-dependent protein kinase II phosphorylation site (CaM peptide), prevents calcium-activated filamin translocation in permeabilized bovine pulmonary artery EC. The myristoylated permeable form of this peptide inhibits bradykinin-induced filamin translocation and F-actin rearrangement in cultured intact ECs. In addition, bradykinin-induced paracellular gap formation is significantly attenuated by CaM peptide, which suggests that the presence of a filamin-based peripheral F-actin network is essential for maintaining EC barrier function. Moreover, CaM peptide reduces wound-induced EC migration rate by 40%, which indicates that F-actin rearrangement is required for efficient cell motility. The CaM peptide affects other bradykinin-induced inflammatory responses. EC nitric oxide synthase (eNOS) translocates from the cell membrane to the nuclear fraction within 1-2 min of bradykinin treatment. Pretreatment with CaM peptide inhibits eNOS translocation. However, the peptide has no effect on bradykinin-induced von Willebrand Factor release. In summary, the CaM peptide exhibits several anti-inflammatory properties that include maintaining EC junctional stability and inhibiting eNOS translocation.
Actins↗
Role of p38 in stress activation of Sp1.
Cell stressors such as physical forces can activate Sp1-dependent genes but the regulatory mechanisms are not defined. We determined if the stress-induced MAP kinase, p38, can phosphorylate Sp1 and thereby regulate the Sp1 target gene FLNA. We used Rat-2 cells and human gingival fibroblasts to examine stress-induced activation of an Sp1-dependent gene and SL2 cells, an Sp1-deficient model system, to facilitate interaction studies of transfected Sp1 with regulatory factors. Mechanical stress applied to Rat-2 cells increased promoter activity of the Sp1 target gene filamin A by >5-fold; activation was blocked by mutations to Sp1 binding sites in the filamin A promoter. Transfection experiments in SL2 cells with Sp1 expression vectors showed that when co-transfected with constitutively active p38, wild-type Sp1 but not an Sp1 binding mutant, increased promoter activity of the Sp1 target gene, filamin A, and enhanced binding of nuclear extracts to a filamin A promoter oligonucleotide. Filamin A promoter activity was blocked by dominant negative p38. Sp1 that was phosphorylated at Thr453 and Thr739 by constitutively active p38 bound to the filamin A promoter more effectively than un-phosphorylated Sp1. Recombinant active p38 phosphorylated wild-type Sp1 in vitro while the Sp1 Thr453Thr739 double mutant protein showed >3-fold reduction of phosphorylation. We conclude that stress activation of p38 phosphorylates Sp1 at specific threonine residues, modifications which in turn enhance the expression of Sp1-dependent genes.
Animals↗
Intracellular distributions of mechanochemical proteins in cultured fibroblasts.
We have used methods that have allowed simultaneous fluorescent staining of intracellular actin together with either myosin, filamin, or tubulin in normal rat kidney fibroblasts in monolayer culture. In the main portions of the cell body, the actin, myosin, and filamin are all present in two structures: in one, the three proteins are present in the same fiber bundles (stress fibers); in the other, there is a diffuse distribution of the three proteins. On portions of the cell periphery however-in the basal regions of microspikes, in ruffles, and in regions of cell-cell contact-actin and filamin are present, but myosin is severely depleted or absent. Microtubules are present in the cell body in a distribution independent of the stress fibers and are mostly absent from the cell periphery. Microspikes and ruffles are highly dynamic structures on the cell surface, and regions of cell-cell contact generally result from the association of ruffles on the two contacting cells. Therefore, the presence of filamin and actin but not myosin in these specialized regions on the cell surface, together with the recent demonstration [Wang, K. & Singer, S. J. (1977) Proc. Natl. Acad. Sci. USA 74, 2021-2025)] that pure filamin interacts with individual F-actin filaments in solution to form fiber bundles and sheet-like structures, suggest that in vivo filamin-actin interactions play an important role in the control of actin filament structure, in cell motility, and in the stabilization of cell-cell contacts.
Cell Line↗
Mutations in two regions of FLNB result in atelosteogenesis I and III.
The filamins are a family of cytoplasmic proteins that bind to and organize actin filaments, link membrane proteins to the cytoskeleton, and provide a scaffold for signaling molecules. Mutations in the gene encoding filamin B (FLNB) cause a spectrum of osteochondrodysplasias, including atelosteogenesis type I (AOI) and atelosteogenesis type III (AOIII). AOI and AOIII are autosomal dominant lethal skeletal dysplasias characterized by overlapping clinical findings that include vertebral abnormalities, disharmonious skeletal maturation, hypoplastic long bones, and joint dislocations. Previous studies have shown that heterozygosity for missense mutations that alter the CH2 domain and repeat 6 region of filamin B produce AOI and AOIII. In this study, 14 novel missense mutations in FLNB were found in 15 unrelated patients with AOI and AOIII. The majority of the mutations resided in exon 2 and exon 3, which encode the CH2 domain of the actin-binding region of filamin B. The remaining mutations were found in exon 28 and exon 29, which encode repeats 14 and 15 of filamin B. These results show that clustering of mutations in two regions of FLNB produce AOI/AOIII, and highlight the important role of this cytoskeletal protein in normal skeletogenesis.
Amino Acid Sequence↗
Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?
The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.
Blood Coagulation↗
Presence of presenilin 1/2 affects the invasion and replication of Salmonella typhimurium.
In this study, we used four different cell lines, with or without presenilin-1 or -2, to investigate the hypothesis that the presence of presenilin, the most prominently mutated gene in Alzheimer's patients, affects the infection rate of host cells by Salmonella. The invasion and replication of Salmonella in presenilin 1/2-deficient cells were significantly lower than those in presenilin 1/2-expressing cells. Among several presenilin-interacting proteins, the expression of filamin-A in presenilin 1/2-deficient cells was significantly lower than in presenilin 1/2-expressing cells. However, Salmonella infection of filamin-A-deficient M2 cells did not significantly differ from infection of filamin-A-containing A7 cells, ruling out the possibility that filamin-A is a major protein inhibiting Salmonella invasion and replication. It is of interest to note that Hes-1 expression, a downstream target of Notch signaling pathway, was significantly decreased by Salmonella infection. Our results demonstrate that the presence of presenilins affects the invasion and replication processes of Salmonella.
Actins↗
Integrin beta cytoplasmic domains differentially bind to cytoskeletal proteins.
Integrin cytoplasmic domains connect these receptors to the cytoskeleton. Furthermore, integrin-cytoskeletal interactions involve ligand binding (occupancy) to the integrin extracellular domain and clustering of the integrin. To construct mimics of the cytoplasmic face of an occupied and clustered integrin, we fused the cytoplasmic domains of integrin beta subunits to an N-terminal sequence containing four heptad repeat sequences. The heptad repeats form coiled coil dimers in which the cytoplasmic domains are parallel dimerized and held in an appropriate vertical stagger. In these mimics we found 1) that both conformation and protein binding properties are altered by insertion of Gly spacers C-terminal to the heptad repeat sequences; 2) that the cytoskeletal proteins talin and filamin are among the polypeptides that bind to the integrin beta1A tail. Filamin, but not talin binding, is enhanced by the insertion of Gly spacers; 3) binding of both cytoskeletal proteins to beta1A is direct and specific, since it occurs with purified talin and filamin and is inhibited in a point mutant (beta1A(Y788A)) or in splice variants (beta1B, beta1C) known to disrupt cytoskeletal associations of beta1 integrins; 4) that the muscle-specific splice variant, beta1D, binds talin more tightly than beta1A and is therefore predicted to form more stable cytoskeletal associations; and 5) that the beta7 cytoplasmic domain binds filamin better than beta1A. The structural specificity of these associations suggests that these mimics offer a useful approach for the analysis of the interactions and structure of the integrin cytoplasmic face.
Amino Acid Sequence↗
Regulation of von Willebrand factor binding to the platelet glycoprotein Ib-IX by a membrane skeleton-dependent inside-out signal.
The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb(alpha) markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.
Animals↗
Biallelic pathogenic variants in FLNB are associated with paediatric steroid-resistant nephrotic syndrome via podocyte cytoskeletal dysfunction.
BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a severe paediatric kidney disease and a leading cause of end-stage kidney disease in children, with a high genetic contribution. While over 80 monogenic causes of SRNS have been identified, a significant proportion of affected patients still lack a clear genetic diagnosis, indicating that additional causative genes remain to be discovered. METHODS: Through whole-exome sequencing of a paediatric SRNS cohort, we identified three probands carrying biallelic FLNB pathogenic variants. Sanger sequencing was performed for familial cosegregation verification and ACMG classification. Expression of Filamin B, Nephrin and Synaptopodin in renal tissues was assessed by immunohistochemistry/immunofluorescence. Wild-type and patient-derived variant FLNB plasmids were constructed and transfected into HEK293T cells and immortalised human podocytes (HPCs). The effects of these variants on protein expression, localisation and cytoskeletal organisation were assessed by western blotting and immunofluorescence. FLNB expression in HPCs was silenced using shRNA to evaluate the impact on podocyte marker proteins, cytoskeletal integrity and migratory capacity. A zebrafish flnb knockdown model was employed to validate its effects on renal development. RESULTS: All three probands presented with isolated SRNS without skeletal developmental abnormalities, and renal tissues showed significantly reduced Filamin B protein expression. In vitro, p.L117P and p.M1803L variants led to markedly reduced protein expression, while p.R470L and p.K2586R induced perinuclear aggregation of Filamin B accompanied by F-actin rearrangement. FLNB silencing led to downregulation of Nephrin and Synaptopodin, cytoskeletal disorganisation and impaired cell migration. Zebrafish flnb knockdown exhibited pericardial oedema, defective nephron development and abnormal podocyte foot processes. CONCLUSION: We report for the first time that biallelic FLNB pathogenic variants are associated with paediatric SRNS by disrupting Filamin B expression, cytoskeletal integrity and podocyte function, providing evidence that FLNB is a novel monogenic cause of SRNS.
Humans↗
Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II.
Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca(2+)-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca(2+)- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.
Animals↗
Differential expression of laminin chains and anti-laminin autoantibodies in experimental lupus nephritis.
Mice with chronic graft-versus-host disease (GvHD) develop a lupus-like disease with severe immune complex glomerulonephritis. Previous studies with this model have shown that anti-laminin autoantibodies are involved in immune complex formation and that glomerular laminin expression alters qualitatively. The present study investigated glomerular laminin chain expression and autoantibody reactivity with matrix antigens during disease development in mice with chronic GvHD, killed before and 6, 8, 10, and 11 weeks after disease induction, using antibodies raised against laminin chain peptides, in immunofluorescence and western blotting studies. Decreased glomerular expression of the laminin beta1 chain, unaltered expression of the laminin beta2 and gamma1 chains, and increased expression of the laminin alpha1 chain and filamin/actin-binding protein 280 (ABP 280) were found during disease progression. Furthermore, 4 weeks after disease induction, autoantibodies appeared which were reactive with laminin alpha1, beta1, beta2, and gamma1 chains, and filamin in rat mesangial cell matrix. Ten weeks after disease induction, autoantibodies reacted with filamin, and beta2 and gamma1 laminin chains. Autoantibodies reacted with laminin chains only and not with other proteins in matrices extracted from glomeruli of normal and diseased mice. Staining with H50, an anti-laminin alpha1 chain/anti-filamin monoclonal autoantibody derived from an MRL/lpr mouse with spontaneous lupus nephritis, confirmed these observations and showed identical anti-laminin/anti-filamin autoantibody reactivity in two different models for lupus nephritis. In summary, differential glomerular expression of laminin chains was found during the development of chronic GvHD. Concomitantly with expression of the laminin alpha1 chain and/or filamin in the glomerulus, anti-laminin alpha1 and/or anti-filamin reactivity was present, pointing towards a role for (neo) antigen expression in the epitope spreading of the immune response. Furthermore, glomerular expression of laminin beta1 decreased in conjunction with decreased presence of anti-laminin beta1 reactivity, presumably due to antigen masking or shedding of immune complexes into the urine. These changes in anti-laminin chain autoantibodies, with concomitant alterations in the glomerular expression of laminin chains, may aggravate progressive immune injury in this model for lupus nephritis.
Animals↗
Hyaluronan-CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells.
In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.
Carcinoma, Squamous Cell↗