RESUMO
FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.
Assuntos
Quinase 1 de Adesão Focal/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/fisiologia , Núcleo Celular/metabolismo , Sobrevivência Celular/fisiologia , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/deficiência , Quinase 1 de Adesão Focal/genética , Mesoderma/patologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estaurosporina/farmacologia , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-alpha (TNFalpha)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK(-/-)) and FAK(+/+) mouse embryo fibroblasts. FAK promoted TNFalpha-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFalpha-mediated nuclear factor-kappaB or c-Jun N-terminal kinase activation. TNFalpha-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK(-/-) fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFalpha-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK(-/-) and A549 FAK short hairpin RNA-expressing cells rescued TNFalpha-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFalpha-stimulated IL-6 expression. Moreover, analyses of Src(-/-), Yes(-/-), and Fyn(-/-) fibroblasts showed that Src expression was inhibitory to TNFalpha-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression.
