RESUMO
Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.
Assuntos
Caspase 3/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Caspase 3/genética , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Microinjeções , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Ovário/citologia , Ovário/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Cultura Primária de Células , Estrutura Terciária de Proteína , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Xenopus laevis , Proteína p120 Ativadora de GTPase/genéticaRESUMO
Beyond its key role in the control of energy metabolism, insulin is also an important regulator of cell division and neoplasia. However, the molecular events involved in insulin-driven cell proliferation are not fully elucidated. Here, we show that the ubiquitin ligase Chfr, a checkpoint protein involved in G2/M transition, is a new effector involved in the control of insulin-induced cell proliferation. Chfr is identified as a partner of the molecular adapter Grb14, an inhibitor of insulin signalling. Using mammalian cell lines and the Xenopus oocyte as a model of G2/M transition, we demonstrate that Chfr potentiates the inhibitory effect of Grb14 on insulin-induced cell division. Insulin stimulates Chfr binding to the T220 residue of Grb14. Both Chfr binding site and Grb14 C-ter BPS-SH2 domain, mediating IR binding and inhibition, are required to prevent insulin-induced cell division. Targeted mutagenesis revealed that Chfr ligase activity and phosphorylation of its T39 residue, a target of Akt, are required to potentiate Grb14 inhibitory activity. In the presence of insulin, the binding of Chfr to Grb14 activates its ligase activity, leading to Aurora A and Polo-like kinase degradation and blocking cell division. Collectively, our results show that Chfr and Grb14 collaborate in a negative feedback loop controlling insulin-stimulated cell division.
Assuntos
Proliferação de Células , Insulina/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Células COS , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Chlorocebus aethiops , Técnicas de Inativação de Genes , Mutagênese , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Xenopus , Quinase 1 Polo-LikeRESUMO
BACKGROUND/AIM: Estrogen-independent breast cancer cell growth is under the control of fibroblast growth factors receptors (FGFRs), but the role of phospholipase C gamma (PLC(gamma)) and Akt, the downstream effectors activated by FGFRs, in cell proliferation is still unresolved. MATERIALS AND METHODS: FGFRs from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a powerful model system to assess the G(2)/M checkpoint regulation. Under FGF1 stimulation, an analysis of the progression in the M-phase of the cell cycle and of the Akt signaling cascades were performed using the phosphatidylinositol-3-kinase inhibitor, LY294002, and a mimetic peptide of the SH3 domain of PLC(gamma). RESULTS: Activated Akt binds and phosphorylates PLC(gamma) before Akt targets the tumor suppressor Chfr. Disruption of the Akt-PLC(gamma) interaction directs Akt binding to Chfr and accelerates the alleviation of the G(2)/M checkpoint. CONCLUSION: The PLC(gamma)-Akt interaction, triggered by FGF receptors from estrogen-independent breast cancer cells MDA-MB-231, regulates progression in the M-phase of the cell cycle.
Assuntos
Neoplasias da Mama/metabolismo , Divisão Celular/fisiologia , Fase G2/fisiologia , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Western Blotting , Eletrofisiologia , Feminino , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Microinjeções , Oócitos/citologia , Oócitos/fisiologia , Transdução de Sinais , Xenopus laevisRESUMO
The Alphavirus Sindbis 6K protein is involved in several functions. It contributes to the processing and membrane insertion of E1 and PE2 viral envelope glycoproteins and to virus budding. It also permeabilizes Escherichia coli and mammalian cells. These viroporin-like properties have been proposed to help virus budding by modifying membrane permeabilities. We expressed Sindbis virus 6K cRNA in Xenopus oocytes to further characterize the effect of 6K on membrane conductances and permeabilization. Although no intrinsic channel properties were seen, cell shrinkage was observed within 24 h. Voltage-clamp experiments showed that 6K upregulated endogenous currents: a hyperpolarization-activated inward current (I (in)) and a calcium-dependent chloride current (I (Cl)). 6K was located at both the plasma and the endoplasmic reticulum membranes. The plasma membrane current upregulation likely results from disruption of the calcium homeostasis of the cell at the endoplasmic reticulum level. Indeed, 6K cRNA expression induced reticular calcium store depletion and capacitative calcium entry activation. By experimental modifications of the incubation medium, we showed that downstream of these events cell shrinkage resulted from a 6K -induced KCl efflux (I (Cl) upregulation leads to chloride efflux, which itself electrically drives potassium efflux), which was responsible for an osmotic water efflux. Our data confirm that 6K specifically triggers a sequential cascade of events that leads to cytoplasmic calcium elevation and cell permeabilization, which likely play a role in the Sindbis virus life cycle.
Assuntos
Alphavirus/fisiologia , Canais de Cálcio/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Oócitos/metabolismo , Proteínas do Envelope Viral/fisiologia , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Feminino , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Xenopus , Xenopus laevisRESUMO
BACKGROUND: Fibroblast growth factors receptors (FGFRs) are involved in estrogen-independent breast cancer cell growth. Grbl4, a member of the Grb7 family of adapters, is an inhibitor of FGFR signaling. MATERIALS AND METHODS: FGFR from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a widely used model system to question cascade transduction regulations. The effect of microinjection of Grb14 and various mimetic peptides for FGFR tyrosine residues were analysed by FGFR immunoprecipitation and Western blot analysis of signaling cascades. RESULTS: PLCy, ERK2, JNK1 and AKT were blocked by Grb14. Only the pY766 phosphopeptide mimetic of the PLCgamma binding site on FGFR released the inhibitory action of Grb14. CONCLUSION: Grb14 binds to the Y766 site of MDA-MB-231-FGFR, competing for PLCy activation, thus inducing an arrest of the signaling transduction cascades.