1,720,974 research outputs found
Meccanismi di regolazione del movimento cellulare: ruolo di tirosin chinasi della famiglia SRC e ABL
No full textIl movimento direzionato è un processo cellulare fondamentale, essenziale per
vari fenomeni biologici e patologici, quali ad esempio l’embriogenesi, la risposta
infiammatoria, la riparazione e lo sviluppo tissutale ed i tumori. È generalmente
accettato che la forza guida per il movimento cellulare è data dalla
riorganizzazione dinamica del citoscheletro di actina, che guida le protrusioni al
fronte della cellula e la ritrazione alla coda, ma è tuttavia noto che un’efficiente
migrazione richiede variazioni ordinate in molte altre attività cellulari, come la
secrezione direzionata, che fornisce nuovi componenti alle membrane, il “turnover”
delle interazioni fra cellula e matrice, che controlla l’adesione, e variazioni nella
trascrizione genica. Perché avvenga quindi un effettivo spostamento della cellula è
necessaria l’integrazione spazio-temporale di numerosi processi, che coinvolgono
il riarrangiamento del citoscheletro di actina e varie proteine accessorie ad esso
associate. Un’efficace migrazione direzionata richiede in particolare l’acquisizione
ed il mantenimento da parte della cellula di un’asimmetria spaziale, che si
manifesta in una morfologia polarizzata, che porta ad una netta distinzione fra il
fronte e la coda della cellula stessa. [...]not availabl
Regulation of phagocyte migration and recruitment by Src-family kinases.
No full textSrc-family kinases (SFKs) regulate different granulocyte and monocyte/macrophage responses. Accumulating evidence suggests that members of this family are implicated in signal transduction pathways regulating phagocytic cell migration and recruitment into inflammatory sites. Macrophages with a genetic deficiency of SFKs display marked alterations in cytoskeleton dynamics, polarization and migration. This same phenotype is found in cells with either a lack of SFK substrates and/or interacting proteins such as Pyk2/FAK, c-Cbl and p190RhoGAP. Notably, SFKs and their downstream targets also regulate monocyte recruitment into inflammatory sites. Depending on the type of assay used, neutrophil migration in vitro may be either dependent on or independent of SFKs. Also neutrophil recruitment in in vivo models of inflammation may be regulated differently by SFKs depending on the tissue involved. In this review we will discuss possible mechanisms by which SFKs may regulate phagocytic cell migratory abilities
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Role of SADDAN-FGFR3 mutant in cytoskeletal disorganization from the endoplasmic reticulum
No full textHuman congenital skeletal dysplasias are caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. FGFR3 is a glycosylated transmembrane tyrosine kinase receptor exposed on the plasma membrane and plays a key role in skeletal development. Mutations associated with severe skeletal dysplasias cause an abnormal intracellular signaling by the receptor. In this study, we analyzed the mutations associated to Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII). In these two disorders, FGFR3 carries the K650 M/E substitutions located in the activation loop of the tyrosine kinase domain. Both substitutions result in a strong ligand-independent constitutive receptor auto-phosphorylation. As previously described, the highly phosphorylated SADDAN and TDII receptors fail to reach full maturation and accumulate in their immature high-mannose-rich isoforms in the endoplasmic reticulum (ER) [1].In this study, we have analyzed whether the FGFR3-SADDAN mutant could affect the cytoskeletal organization by altering actin fibers through paxillin activation. Paxillin is a multidomain focal adhesion-associated protein that plays a key role in integrin signaling and in the control of cytoskeletal organization, thus exerting a central role in the regulation of cell morphology. A critical site for paxillin activation is phosphorylation of Tyrosine (Tyr) 118 through FAK (Focal Adhesion Kinase) and Src proteins.By immunoprecipitation (IP) analysis with specific anti-paxillin antibodies, we assayed whether the phosphorylation of paxillin at Tyr 118 was induced by the SADDAN receptor. For this purpose, we have performed transient transfection in HEK293 cells with plasmids carrying the murine FGFR3-wt-HA, SADD-HA, TDII-HA and other FGFR3 mutant cDNAs. We observed that the level of paxillin phosphorylation at Tyr 118 was highly induced by the FGFR3-SADDAN. Interestingly, the TDII mutant, although highly auto-phosphorylated, it did not increase the paxillin phosphorylation level, suggesting that paxillin is a specific target of SADDAN-FGFR3.To investigate on a possible interaction between paxillin and SADDAN-FGFR3, we have performed co-immunoprecipitation experiments. Our data indicate that paxillin does not co-immunoprecipitate the FGFR3 mutant. The FGFR3-SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not affect paxillin phosphorylation at Tyr-118, suggesting that the enzymatic activity is required for such activation. Interestingly, the double mutant SADDAN-754, abolishing the binding with PLC-γ1, an effector of FGFR3 signaling from the ER [2], did not affect paxillin phosphorylation, suggesting a role for this protein in paxillin activation. Furthermore, by confocal analysis, we showed in HeLa cells that the actin stress fibers are disorganized in cells expressing the FGFR3-SADDAN, but not in cells expressing wild-type FGFR3 or TDII receptors. In addition, our analysis reveals that paxillin is organized in peripheral area in FGFR3-wt cells while in SADDAN cells paxillin is distributed through the cytoplasm. A further observation is that phospho-paxillin co-localizes with FGFR3-SADDAN mutant but not with FGFR3-wt.In conclusion we show evidences that paxillin is associated with the ER and strongly phosphorylated by FGFR3-SADDAN. Our future studies will be focused on determining the paxillin interaction with other structural and signaling proteins of the focal adhesion structures (such as FAK and Src) to reveal the molecular events leading to actin cytoskeletal disorganization by FGFR3-SADDAN.[1] Lievens, P. M-J., Mutinelli, C., Baynes, D. & Liboi, E. The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling. J. Biol. Chem. 279, 43254–43260 (2004).[2] Lievens, P. M.-J., Roncador, A., and Liboi, E. K644E/M FGFR3 Mutants Activate Erk1/2 from the Endoplasmic Reticulum through FRS2a and PLCg-independent Pathways. J. Mol. Biol. 357, 783–792 (2006)
Abnormal signaling by FGFR3-K650M mutant associated with SADDAN disease targets paxillin causing cytoskeleton disorganization.
No full textMutations in the fibroblast growth factor receptor 3 (FGFR3) gene cause chondrodysplasias. Change of lysine 650 to methionine (K650M) leads to Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN), a disease characterized by constitutive FGFR3 tyrosine kinase activity. We previously showed that autophosphorylation occurs early during protein biosynthesis, hampering complete maturation of SADDAN receptors. A similar behaviour was observed when lysine 650 was sustituted by glutamic acid (K650E), a mutation associated with Thanatophoric Dysplasia type II (TDII). Both mutants accumulate in the ER/Golgi as highly phosphorylated immature receptors from where they trigger an abnormal signalling. Based on the observation that SADDAN-FGFR3 causes alterations in cell morphology, we analyzed whether this mutant could affect cytoskeletal organization through paxillin (PXN) function. PXN is a focal adhesion-associated protein playing a key role in cytoskeletal organization. A critical event for PXN activation is phosphorylation at tyrosine 118 by FAK and Src proteins. Our data show that SADDAN-FGFR3 enhanced PXN phosphorylation at tyrosine 118 causing cell morphology changes. The SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not change PXN phosphorylation, implying the requirement of receptor enzymatic activity. Interestingly, the kinase active TDII-FGFR3, had no effect on PXN phosphorylation, suggesting that PXN is a specific target of SADDAN-FGFR3. Finally, the SADDAN-Y760F mutant abolishing the interaction with PLCγ1 rescued the effect on PXN, suggesting a role for PLCγ1 in PXN activation.The results of this study will contribute to clarify the molecular events leading to actin cytoskeletal disorganization by SADDAN-FGFR3
SADDAN-FGFR3 signalling involves paxillin phosphorylation and causes cytoskeleton disorganization.
No full textMutations in the fibroblast growth factor receptor 3 (FGFR3) gene cause chondrodysplasias. FGFR3 is a tyrosine kinase (TK) receptor playing a key role in skeletal development as a negative regulator of bone growth. In this study, we analyzed gain-of-function mutations associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII). In these two severe dwarfisms, FGFR3 carries the K650M and K650E substitutions, respectively, located in the activation loop of the TK-domain. Both substitutions result in a strong ligand-independent constitutive FGFR3 activation. The highly phosphorylated SADDAN and TDII receptors fail to reach full maturation and accumulate in their immature high mannose-rich forms in the endoplasmic reticulum, from where they induce abnormal signalling. We analyzed whether the SADDAN-FGFR3 mutant could affect cytoskeletal organization through paxillin (PXN) activation. PXN is a focal adhesion-associated protein playing a key role in cytoskeletal organization and cell morphology regulation. A critical site for PXN activation is phosphorylation at tyrosine (Tyr) 118 through FAK and Src proteins. Our data show that SADDAN-FGFR3 enhances PXN phosphorylation at Tyr-118 and causes cell morphology changes. The SADDAN-KD (kinase dead) mutant, lacking kinase activity, did not affect PXN phosphorylation, indicating the requirement of receptor enzymatic activity. Similar results were obtained with the SADDAN-Y754F double mutant abolishing the interaction with PLCγ1, suggesting a role for this effector in PXN activation. Interestingly, the TDII-FGFR3 mutant, although highly auto-phosphorylated, did not increase PXN phosphorylation, suggesting that PXN is a specific target of SADDAN-FGFR3. The results of this study will contribute to clarify the molecular events leading to actin cytoskeletal disorganization by SADDAN-FGFR3
SADDAN-FGFR3 causes cytoskeleton disorganization and paxillin hyperphosphorylation by Src
No full textchondrodysplasias. FGFR3 is a tyrosine kinase (TK) receptor playing a key role in skeletal development. In this study, we analyzed mutations of FGFR3 associated with Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN) and with Thanatophoric Dysplasia type II (TDII), carrying the K650M and K650E substitutions, respectively. Both substitutions affect the TK-domain functions, resulting in a strong ligand-independent constitutive FGFR3 activation. The highly phosphorylated SADDAN and TDII receptors fail to reach full maturation and accumulate in their immature isoforms in the endoplasmic reticulum, from where they induce abnormal signalling. Objective: This study aimed to investigate whether the SADDAN-FGFR3 signalling could affect cytoskeletal organization through paxillin phosphorylation. Paxillin is a focal adhesion-associated protein playing an important role in cytoskeletal organization, cell morphology regulation, migration and proliferation. Paxillin is phosphorylated at Tyr118 by FAK and Src proteins. Methods: Paxillin phosphorylation was analyzed in HEK293 cells expressing FGFR3 mutants receptors by immunoprecipitation with anti-paxillin antibodies and immunoblotting with anti-phospho-paxillin (Tyr118) antibodies. Cytoskeletal changes and paxillin localization were analyzed in HeLa cells by immunofluorescence. Results: SADDAN-FGFR3 enhances paxillin phosphorylation at Tyr118 causing cell morphology alterations, and partially colocalizes with phosphorylated paxillin. The SADDAN-KD mutant, lacking kinase activity, does not affect paxillin phosphorylation, indicating the requirement of receptor enzymatic activity. Conversely, the TDII-FGFR3 mutant, although highly auto-phosphorylated, does not affect paxillin phosphorylation. Interestingly, PLC-γ1 plays a key role in paxillin hyperphosphorylation since the SADDAN-Y754F double mutant, abolishing the binding to PLC-γ1, does not enhance paxillin phosphorylation. Finally, the Src kinases inhibitor PP2 downregulates paxillin hyperphosphorylation, suggesting a role for Src in paxillin phospho-alterations.Conclusions: Paxillin is recruited by SADDAN-FGFR3 and hyperphosphorylated through Src kinases. The results of this study will contribute to clarify the molecular events leading to actin cytoskeletal disorganization by SADDAN-FGFR3
The SRC family kinases hck and fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine.
No full textThe chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization
Sos1 regulates macrophage podosome assembly and macrophage invasive capacity
No full textPodosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation
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