RESUMEN
Diverse G protein-coupled receptors depend on Gßγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gßγ. Thus, we tested the hypothesis that Gßγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gßγ. Cell stimulation with serum modulates Gßγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gßγ heterodimers containing different Gß subunits, except Gß4. Both, mTORC1 and mTORC2 complexes interact with Gß1γ2 which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gßγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gßγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gßγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gßγ signaling interfaces.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Western Blotting , Activación Enzimática/efectos de los fármacos , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Humanos , Inmunoprecipitación , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The mammalian target of rapamycin, best known as mTOR, is a phylogenetically conserved serine/threonine kinase that controls life-defining cellular processes such as growth, metabolism, survival, and migration under the influence of multiple interacting proteins. Historically, the cellular activities blocked by rapamycin in mammalian cells were considered the only events controlled by mTOR. However, this paradigm changed with the discovery of two signaling complexes differentially sensitive to rapamycin, whose catalytic component is mTOR. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to growth factors and nutrients via the phosphorylation of p70S6K and 4EBP1; while the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of AKT, respectively. Although mTORC2 kinase activity is not inhibited by rapamycin, hours of incubation with this antibiotic can impede the assembly of this signaling complex. The direct mechanism by which mTORC2 leads to cell migration depends on its interaction with P-Rex1, a Rac-specific guanine nucleotide exchange factor, while additional indirect pathways involve the intervention of PKC or AKT, multifunctional ubiquitous serine/threonine kinases that activate effectors of cell migration upon being phosphorylated by mTORC2 in response to chemotactic signals. These mTORC2 effectors are altered in metastatic cancer. Numerous clinical trials are testing mTOR inhibitors as potential antineoplasic drugs. Here, we briefly review the actions of mTOR with emphasis on the controlling role of mTORC1 and mTORC2-interacting proteins and highlight the mechanisms linked to cell migration.
Asunto(s)
Metabolismo Energético/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Sirolimus/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/metabolismo , Movimiento Celular/fisiología , Proliferación Celular , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos MultiproteicosRESUMEN
G-protein coupled receptors activate heterotrimeric G proteins at the plasma membrane in which most of their effectors are intrinsically located or transiently associated as the external signal is being transduced. This paradigm has been extended to the intracellular compartments by studies in yeast showing that trafficking of Galpha activates phosphatidylinositol 3-kinase (PI3K) at endosomal compartments, suggesting that vesicle trafficking regulates potential actions of Galpha and possibly Gbetagamma at the level of endosomes. Here, we show that Gbetagamma interacts with Rab11a and that the two proteins colocalize at early and recycling endosomes in response to activation of lysophosphatidic acid (LPA) receptors. This agonist-dependent association of Gbetagamma to Rab11a-positive endosomes contributes to the recruitment of PI3K and phosphorylation of AKT at this intracellular compartment. These events are sensitive to the expression of a dominant-negative Rab11a mutant or treatment with wortmannin, suggesting that Rab11a-dependent Gbetagamma trafficking promotes the activation of the PI3K/AKT signaling pathway associated with endosomal compartments. In addition, RNA interference-mediated Rab11a depletion, or expression of a dominant-negative Rab11a mutant attenuated LPA-dependent cell survival and proliferation, suggesting that endosomal activation of the PI3K/AKT signaling pathway in response to Gbetagamma trafficking, via its interaction with Rab11, is a relevant step in the mechanism controlling these fundamental events.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Endosomas/metabolismo , Humanos , Microscopía Fluorescente/métodos , Modelos Biológicos , Mutación , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
OBJECTIVE: Little is known about the causes of sporadic (noninherited) retinoblastoma. Rates seem to be somewhat higher among poorer populations in Mexico. Fruits and vegetables are important sources of carotenoids and folate. We examined whether decreased gestational maternal intake of fruits and vegetables may contribute to development of sporadic retinoblastoma. METHODS: At the Instituto Nacional de Pediatria in Mexico City, we conducted a hospital-based case-control study to evaluate prenatal maternal diet. We examined dietary intake of fruits and vegetables of mothers of 101 children with retinoblastoma and 172 control children using a dietary recall questionnaire and published food nutrient content tables. RESULTS: The reported number of mean daily servings of fruits and vegetables was lower among case mothers when compared with control mothers [vegetables: 2.28 in controls, 1.75 in cases (P < 0.01); fruits: 2.13 in controls, 1.59 in cases (P = 0.07)]. Mean daily maternal folate intake from both vegetables and fruits was higher in controls (103 microg) than in cases (48 microg; P < 0.05). Risk for having a child with retinoblastoma was increased for mothers consuming fewer than 2 daily servings of vegetables [odds ratios (OR), 3.4; 95% confidence interval (95% CI), 2.0-6.0] or with a low intake of folate (OR, 3.9; 95% CI, 2.1, 7.3), or lutein/zeaxanthin (OR, 2.6; 95% CI, 1.5-4.6) derived from fruits and vegetables. CONCLUSIONS: Decreased intake of vegetables and fruits during pregnancy and the consequent decreased intake of nutrients such as folate and lutein/zeaxanthin, necessary for DNA methylation, synthesis, and retinal function, may increase risk for having a child with sporadic retinoblastoma.