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See detailThe actin filament cross-linker L-plastin confers resistance to TNF-alpha in MCF-7 breast cancer cells in a phosphorylation-dependent manner.
Janji, Bassam; Vallar, Laurent; Al Tanoury, Ziad et al

in Journal of Cellular & Molecular Medicine (2010), 14(6A), 1264-75

We used a tumour necrosis factor (TNF)-alpha resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine ... [more ▼]

We used a tumour necrosis factor (TNF)-alpha resistant breast adenocarcinoma MCF-7 cell line to investigate the involvement of the actin cytoskeleton in the mechanism of cell resistance to this cytokine. We found that TNF resistance correlates with the loss of cell epithelial properties and the gain of a mesenchymal phenotype, reminiscent of an epithelial-to-mesenchymal transition (EMT). Morphological changes were associated with a profound reorganization of the actin cytoskeleton and with a change in the repertoire of expressed actin cytoskeleton genes and EMT markers, as revealed by DNA microarray-based expression profiling. L-plastin, an F-actin cross-linking and stabilizing protein, was identified as one of the most significantly up-regulated genes in TNF-resistant cells. Knockdown of L-plastin in these cells revealed its crucial role in conferring TNF resistance. Importantly, overexpression of wild-type L-plastin in TNF-sensitive MCF-7 cells was sufficient to protect them against TNF-mediated cell death. Furthermore, we found that this effect is dependent on serine-5 phosphorylation of L-plastin and that non-conventional protein kinase C isoforms and the ceramide pathway may regulate its phosphorylation state. The protective role of L-plastin was not restricted to TNF-alpha resistant MCF-7 cells because a correlation between the expression of L-plastin and the resistance to TNF-alpha was observed in other breast cancer cell lines. Together, our study discloses a novel unexpected role of the actin bundling protein L-plastin as a cell protective protein against TNF-cytotoxicity. [less ▲]

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See detailQuantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth UL; Halavatyi, Aliaksandr UL et al

in PloS one (2010), 5(2), 9210

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers ... [more ▼]

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion. [less ▲]

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See detailOverexpression of the partially activated alpha(IIb)beta3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet-like extensions in Chinese hamster ovary cells
Schaffner-Reckinger, Elisabeth UL; Salsmann, Alexandre UL; Debili, Najet et al

in Journal of Thrombosis and Haemostasis [=JTH] (2009), 7(7), 1207-1217

BACKGROUND: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited ... [more ▼]

BACKGROUND: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. RESULTS: Here we demonstrate that the partially activated alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. CONCLUSIONS: CHO alpha(IIb)beta3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions. [less ▲]

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See detailA nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia
Ghevaert, Cedric; Salsmann, Alexandre UL; Watkins, Nicholas A. et al

in Blood (2008), 111(7), 3407-3414

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin ... [more ▼]

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree. [less ▲]

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See detailThe talin rod IBS2 alpha-helix interacts with the beta3 integrin cytoplasmic tail membrane-proximal helix by establishing charge complementary salt bridges.
Rodius, Sophie; Chaloin, Olivier; Moes, Michèle UL et al

in The Journal of biological chemistry (2008), 283(35), 24212-23

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a ... [more ▼]

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin beta subunits to the cytoskeleton ( Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280-17288 ). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3-talin rod interaction relies on a helix-helix association between alpha-helix 50 of the talin rod domain and the membrane-proximal alpha-helix of the beta3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation. [less ▲]

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See detailRGD, the Rho'd to cell spreading
Salsmann, Alexandre UL; Schaffner-Reckinger, Elisabeth UL; Kieffer, Nelly UL

in European Journal of Cell Biology (2006), 85(3-4), 249-254

Some RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding. However, the precise involvement of each of these recognition sites during cell adhesion is ... [more ▼]

Some RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding. However, the precise involvement of each of these recognition sites during cell adhesion is still unclear. Here we review recent investigations on integrin alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen providing evidence that the fibrinogen synergy gamma(400-411) sequence by itself promotes cell attachment by initiating alphaIIbbeta3 clustering and recruitment of intracellular proteins to focal complexes, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to induce a conformational change necessary for RhoA activation and full cell spreading. [less ▲]

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See detailA new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading
Salsmann, Alexandre UL; Schaffner-Reckinger, Elisabeth UL; Kabile, Fabrice UL et al

in Journal of Biological Chemistry (2005), 280(39), 33610-33619

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function ... [more ▼]

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2. [less ▲]

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See detailThe effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function.
Joutsi-Korhonen, Lotta; Preston, Sandy; Smethurst, Peter A. et al

in Thrombosis and haemostasis (2004), 91(4), 743-54

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of ... [more ▼]

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy. [less ▲]

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See detailBeta3 integrins: major therapeutic targets of the near future.
Schaffner-Reckinger, Elisabeth UL

in Bulletin de la Société des Sciences Médicales du Grand-Duché de Luxembourg (2003), (1), 23-34

Integrins are major cell adhesion receptors which assume two important functions: first they act as anchoring molecules allowing firm cellular attachment to the extracellular matrix, and second they work ... [more ▼]

Integrins are major cell adhesion receptors which assume two important functions: first they act as anchoring molecules allowing firm cellular attachment to the extracellular matrix, and second they work as signalling receptors being able to transduce signals in both directions (outside-in and inside-out) through the plasma membrane. Their biological importance is determined by their involvement in many physiological phenomena. Furthermore, their implication in various diseases and their accessibility to drugs make them interesting therapeutic targets. [less ▲]

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See detailHPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA-1a epitope.
Watkins, Nicholas A.; Schaffner-Reckinger, Elisabeth UL; Allen, David L. et al

in Blood (2002), 99(5), 1833-9

A single nucleotide polymorphism (SNP) at position 196 in the beta 3 integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the beta 3Leu33 form (human platelet antigen ... [more ▼]

A single nucleotide polymorphism (SNP) at position 196 in the beta 3 integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the beta 3Leu33 form (human platelet antigen [HPA]-1a, Pl(A1), Zw(a)) in patients who are beta 3Pro33 homozygous (HPA-1b1b, Pl(A2A2), Zw(bb)) causes neonatal alloimmune thrombocytopenia, posttransfusion purpura, or refractoriness to platelet transfusion. Studies with recombinant proteins have demonstrated that amino acids 1 to 66 and 288 to 490 of the beta 3 integrin contribute to HPA-1a epitope formation. In determining the HPA-1a status of more than 6000 donors, we identified a donor with an HPA-1a(weak) phenotype and an HPA-1a1b genotype. The platelets from this donor had normal levels of surface alpha IIb beta 3 but reacted only weakly with monoclonal and polyclonal anti-HPA-1a by whole blood enzyme-linked immunosorbent assay (ELISA), flow cytometry, and sandwich ELISA. We reasoned that an alteration in the primary nucleotide sequence of the beta 3Leu33 allele of this donor was disrupting the HPA-1a epitope. In agreement with this hypothesis, sequencing platelet RNA-derived alpha IIb and beta 3 cDNA identified a novel G/A SNP at position 376 of the beta 3 integrin that encodes for an Arg93Gln replacement in the beta 3Leu33 allele. Coexpression of the beta 3Leu33Gln93 encoding cDNA in Chinese hamster ovary cells with human alpha IIb cDNA showed that the surface-expressed alpha IIb beta 3 reacted normally with beta 3 integrin-specific monoclonal antibodies but only weakly with monoclonal anti-HPA-1a. Our results show that an Arg93Gln mutation in the beta 3Leu33 encoding allele disrupts the HPA-1a epitope, suggesting that Arg93 contributes to the formation of the HPA-1a B-cell epitope. [less ▲]

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See detailGreen fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.
Plançon, Sébastien UL; Morel-Kopp, M. C.; Schaffner-Reckinger, Elisabeth UL et al

in The Biochemical journal (2001), 357(Pt 2), 529-36

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either ... [more ▼]

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions. [less ▲]

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See detailEvidence from site-directed mutagenesis that the cytoplasmic domain of the beta3 subunit influences the conformational state of the alphaVbeta3 integrin ectodomain.
Schaffner-Reckinger, Elisabeth UL; Brons, N. H.; Kieffer, N.

in Thrombosis and haemostasis (2001), 85(4), 716-23

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes ... [more ▼]

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on beta3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire beta3 cytoplasmic domain induced constitutive LIBS exposure on alphavbeta3 and alphaIIbeta3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on alphavbeta3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the beta3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the alphavbeta3 ectodomain, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes. [less ▲]

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See detailDistinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.
Schaffner-Reckinger, Elisabeth UL; Gouon, V.; Melchior, Chantal UL et al

in The Journal of biological chemistry (1998), 273(20), 12623-32

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3 ... [more ▼]

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events. [less ▲]

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