RESUMO
Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells.
RESUMO
The Akt kinase is a critical effector in growth factor signaling. Activation of Akt driven by the growth factor dependent PI3K (phosphatidylinositol-3-OH kinase) is coupled to the plasma membrane translocation and phosphorylation of Akt on two sites by PDK1 (phosphoinositide-dependent protein kinase-1) on Thr-308 and by mTORC2 (mammalian Target of Rapamycin Complex 2) on Ser-473. In our study we examined the sub-cellular localization of mTORC2 and identified that this kinase complex predominantly resides on endoplasmic reticulum (ER). Our immunostaining analysis did not show a substantial co-localization of the mTORC2 component rictor with Golgi, lysosome, clathrin-coated vesicles, early endosomes, or plasma membrane but indicated a strong co-localization of rictor with ribosomal protein S6 and ER marker. Our biochemical study also identified the mTORC2 components rictor, SIN1, and mTOR as the highly abundant proteins in the ER fraction, whereas only small amount of these proteins are detected in the plasma membrane and cytosolic fractions. We found that growth factor signaling does not alter the ER localization of mTORC2 and also does not induce its translocation to the plasma membrane. Based on our study we suggest that the mTORC2-dependent phosphorylation of Akt on Ser-473 takes place on the surface of ER.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas de Transporte/análise , Linhagem Celular , Membrana Celular/enzimologia , Vesículas Revestidas por Clatrina/enzimologia , Retículo Endoplasmático/química , Endossomos/enzimologia , Complexo de Golgi/enzimologia , Humanos , Imuno-Histoquímica , Lisossomos/enzimologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Coloração e Rotulagem , Serina-Treonina Quinases TOR/análiseRESUMO
Cells have been described under the microscope as organelles containing cytoplasm and the nucleus. However, an unnoted structure exists between the cytoplasm and the nucleoplasm of eukaryotic cells. In addition to the nuclear envelope, there exists a perinuclear region (PNR or perinucleus) with unknown composition and function. Until now, an investigation of the role of the perinucleus has been restricted by the absence of a PNR isolation method. This manuscript describes a perinucleus isolation technique on the basis of its unique compact organization. The perinucleus was found to contain approximately 15 to 18% of the total proteins of the mammalian cell, almost half of the proteins of nuclei. Using four different normal and cancer cell lines, it was shown that the composition of PNR is highly dynamic. Application of the method showed that translocation of the p53 tumor-suppressor protein to the perinucleus in immortalized MEF cells is correlated with the translocation of p53-stabilizing protein, nucleophosmin (B23), to the PNR. Herein, the concept of the perinuclear region is advanced as a formal, identifiable structure. The roles of the perinucleus in maintaining genome integrity, regulation of gene expression and understanding of malignant transformation are discussed.
Assuntos
Núcleo Celular/metabolismo , Células/citologia , Células/metabolismo , Citoplasma/metabolismo , Diferenciação Celular , Linhagem Celular , Transformação Celular Neoplásica , Células/patologia , Citosol/metabolismo , Humanos , Modelos Biológicos , Transporte Proteico , Transdução de Sinais , Frações SubcelularesRESUMO
Phosphorylation and activation of Akt1 is a crucial signaling event that promotes adipogenesis. However, neither the complex multistep process that leads to activation of Akt1 through phosphorylation at Thr³°8 and Ser47³ nor the mechanism by which Akt1 stimulates adipogenesis is fully understood. We found that the BSD domain-containing signal transducer and Akt interactor (BSTA) promoted phosphorylation of Akt1 at Ser47³ in various human and murine cells, and we uncovered a function for the BSD domain in BSTA-Akt1 complex formation. The mammalian target of rapamycin complex 2 (mTORC2) facilitated the phosphorylation of BSTA and its association with Akt1, and the BSTA-Akt1 interaction promoted the association of mTORC2 with Akt1 and phosphorylation of Akt1 at Ser47³ in response to growth factor stimulation. Furthermore, analyses of bsta gene-trap murine embryonic stem cells revealed an essential function for BSTA and phosphorylation of Akt1 at Ser47³ in promoting adipocyte differentiation, which required suppression of the expression of the gene encoding the transcription factor FoxC2. These findings indicate that BSTA is a molecular switch that promotes phosphorylation of Akt1 at Ser47³ and reveal an mTORC2-BSTA-Akt1-FoxC2-mediated signaling mechanism that is critical for adipocyte differentiation.
Assuntos
Adipócitos/fisiologia , Adipogenia/fisiologia , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
The mammalian Target Of Rapamycin (mTOR) protein is a central component of the essential and highly conserved signaling pathway that emerged as a critical effector in regulation of cell physiology. Biochemical studies defined mTOR as the protein kinase that exists at least in two distinct complexes. The first complex has been characterized as the nutrient-sensitive mTOR complex 1 that controls cell growth and cell size by regulating protein synthesis and autophagy. The second complex of mTOR has been defined as the component of growth factor signaling that functions as a major regulatory kinase of Akt/PKB. Here, we provide the detailed methods how to purify the functional complexes of mTOR by affinity purification. In the first part, we describe the purification of the distinct mTOR complexes by immunoprecipitation. Purification of the soluble mTOR complexes is explained in the second part of this chapter.