RESUMO
Fgr participates in integrin signaling in myeloid leukocytes. To examine the role of its specific domains in regulating cell migration, we expressed various Fgr molecules in COS-7 cells. Full-length, membrane-bound Fgr, but not an N-terminal truncation mutant that distributed to an intracellular compartment, increased cell migration on fibronectin and enhanced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K), cortactin and focal adhesion kinase (FAK) at Y397 and Y576. Fgr increased Rac GTP loading, and phosphorylation of the Rac GEF Vav2, and bound to a protein complex formed by the Rho inhibitor p190RhoGAP and FAK, increasing p190RhoGAP phosphorylation, in a manner absolutely dependent on membrane localization. A kinase-defective truncation mutant of Fgr increased cell migration, albeit to a much lower extent than full-length Fgr, and was found to associate with the plasma membrane, to activate Rac and to form complexes with p190RhoGAP/FAK. Formation of complexes between p190RhoGAP, Fgr, and the FAK-related protein Pyk2 were also detected in murine macrophages. These findings suggest that the proto-oncogene Fgr regulates cell migration impinging on a signaling pathway implicating FAK/Pyk2 and leading to activation of Rac and the Rho inhibitor p190RhoGAP.
Assuntos
Membrana Celular/metabolismo , Movimento Celular/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Células COS , Chlorocebus aethiops , Cortactina , Ativação Enzimática , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Macrófagos/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Testes de Precipitina , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família srcRESUMO
Primary macrophages isolated from hck(-/-)fgr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-)fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-)fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-)fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-)fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-)fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway.
Assuntos
Actinas/metabolismo , Quimiotaxia de Leucócito/fisiologia , Macrófagos/metabolismo , Proteínas Oncogênicas de Retroviridae/deficiência , Actinas/efeitos dos fármacos , Animais , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteína Oncogênica v-cbl , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck , Proteínas Oncogênicas de Retroviridae/efeitos dos fármacos , Proteínas Oncogênicas de Retroviridae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Tirosina/metabolismo , Quinases da Família src/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
Using a mouse model of allergic lung inflammation, we found that mice deficient of Fgr, a Src family tyrosine kinase highly expressed in myelomonocytic cells, fail to develop lung eosinophilia in response to repeated challenge with aerosolized OVA. Both tissue and airway eosinophilia were markedly reduced in fgr(-/-) mice, whereas mice with the sole deficiency of Hck, another Src family member, responded normally. Release of allergic mediators, such as histamine, IL-4, RANTES/CCL5, and eotaxin/CCL11, in the airways of OVA-treated animals was equal in wild-type and fgr(-/-) mice. However, lung eosinophilia in Fgr-deficient mice correlated with a defective accumulation of GM-CSF and IL-5 in the airways, whereas secretion of these cytokines by spleen cells in response to OVA was normal. Examination of mRNA expression in whole lung tissue allowed us to detect comparable expression of transcripts for eotaxin/CCL11, macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4, monocyte chemoattractant protein-1/CCL2, TCA-3/CCL1, IL-4, IL-10, IL-2, IL-3, IL-9, IL-15, and IFN-gamma in OVA-sensitized wild-type and fgr(-/-) mice. In contrast, the increase in IL-5 and IL-13 mRNA expression was lower in fgr(-/-) compared with wild-type mice. These findings suggest that deficiency of Fgr results in a marked reduction of lung eosinophilia and the establishment of a positive feedback loop based on autocrine secretion of eosinophil-active cytokines. These results identify Fgr as a novel pharmacological target to control allergic inflammation.