RESUMO
On phosphorylation of Cbl, the c-Cbl-associated protein (CAP)/Cbl complex dissociates from the insulin receptor and translocates to a lipid raft membrane fraction to form a ternary complex with flotillin. Deletion analyses of the CAP gene identified a 115-aa region responsible for flotillin binding. This region is homologous to the peptide sorbin and is referred to as the sorbin homology (SoHo) domain. This domain is present in two other proteins, vinexin and ArgBP2. Vinexin also interacted with flotillin, and deletion of its SoHo domain similarly blocked flotillin binding. The overexpression of a CAP mutant in which the SoHo domain had been deleted (CAPDeltaSoHo) prevented the translocation of Cbl to lipid rafts and subsequently blocked the recruitment of CrkII and C3G. Moreover, overexpression of CAPDeltaSoHo prevented the stimulation of glucose transport and GLUT4 translocation by insulin. These results suggest a mechanism for localization of signaling proteins to the lipid raft that mediates the compartmentalization of crucial signal transduction pathways.
Assuntos
Metabolismo dos Lipídeos , Peptídeos/química , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Células 3T3 , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Glucose/metabolismo , Humanos , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-cbl , Homologia de Sequência de AminoácidosRESUMO
The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface. Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation. We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP. On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin. Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.
Assuntos
Adipócitos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Ubiquitina-Proteína Ligases , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Ativação Enzimática , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas c-crk , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
GH signals by interacting with GH receptor (GHR). A substantial fraction of circulating GH complexes with GH-binding protein (GHBP), which corresponds to the GHR extracellular domain. GHBP is generated by 1) alternative splicing of a common GHR precursor messenger RNA to encode secreted GHBP (the source of the vast majority of GHBP in rodents); and 2) proteolysis of the cell-associated GHR with shedding of GHBP (a mechanism operative in rabbits and humans). We previously observed that phorbol ester (PMA)-induced activation of protein kinase C (PKC) causes metalloprotease-mediated GHR proteolysis and GHBP shedding in human IM-9 lymphocytes. We now demonstrate that PMA-induced hydroxamate (IC3)-inhibitable GHR proteolysis and GHBP shedding were also detected in murine 3T3-F442A and 3T3-L1 preadipocytes and in Chinese hamster ovary (CHO) cells stably expressing rabbit GHR (rbGHR), although the degree of GHBP shedding was much smaller for murine GHR than for rabbit or human GHRs. PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells was significantly reduced by pretreatment with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitors, suggesting involvement of the mitogen-activated protein kinase pathway in regulating this PKC-dependent effect. In contrast, GHR proteolysis promoted by N-ethylmaleimide, although inhibited by IC3, was unaffected by inhibition of either PKC or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. Thus, different pathways leading to metalloprotease-mediated receptor proteolysis are accessed by PMA vs. N-ethylmaleimide. To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we tested effects of platelet-derived growth factor (PDGF) and serum. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promoted GHR proteolysis, which was inhibited by IC3. Interestingly, PMA-, PDGF-, and serum-induced GHR proteolysis was associated with substantial decreases in GH-induced activation of Janus kinase-2, which were also prevented by IC3. These findings suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.
Assuntos
Proteínas de Transporte/metabolismo , Substâncias de Crescimento/farmacologia , Peptídeo Hidrolases/metabolismo , Receptores da Somatotropina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células 3T3 , Adipócitos/metabolismo , Animais , Células CHO , Cricetinae , Regulação para Baixo , Etilmaleimida/farmacologia , Ácidos Hidroxâmicos/farmacologia , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Coelhos , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , TransfecçãoRESUMO
In adipose and muscle, insulin stimulates glucose uptake and glycogen synthase activity. Phosphatidylinositol 3-kinase (PI3K) activation is necessary but not sufficient for these metabolic actions of insulin. The insulin-stimulated translocation of phospho-c-Cbl to lipid rafts, via its association with CAP, comprises a second pathway regulating GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of a dominant negative CAP mutant (CAP Delta SH3) completely blocked the insulin-stimulated glucose transport and glycogen synthesis but only partially inhibited glycogen synthase activation. In contrast, CAP Delta SH3 expression did not affect glycogen synthase activation by insulin in the absence of extracellular glucose. Moreover, CAP Delta SH3 has no effect on the PI3K-dependent activation of protein phosphatase-1 or phosphorylation of glycogen synthase kinase-3. These results indicate blockade of the c-Cbl/CAP pathway directly inhibits insulin-stimulated glucose uptake, which results in secondary inhibition of glycogen synthase activation and glycogen synthesis.
Assuntos
Adipócitos/metabolismo , Glicogênio Sintase/metabolismo , Insulina/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Células 3T3 , Animais , Ativação Enzimática , Glucose/metabolismo , Glicogênio/biossíntese , Camundongos , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Domínios de Homologia de srcRESUMO
Insulin resistance is thought to be the primary defect in the pathophysiology of type 2 diabetes. Thus, understanding the cellular mechanisms of insulin action may contribute significantly to developing new treatments for this disease. Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction for the hormone. One potential clue to understanding the unique cellular effects of insulin may lie in the compartmentalization of signaling molecules and metabolic enzymes. We review this evidence, and speculate on how PI-3 kinase-independent and -dependent signaling pathways both diverge from the insulin receptor and converge at discrete targets to insure the specificity of insulin action.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Insulina/fisiologia , Proteínas Musculares , Transdução de Sinais/fisiologia , Animais , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Humanos , Resistência à Insulina , Metabolismo dos Lipídeos , Modelos Biológicos , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor de Insulina/fisiologiaRESUMO
Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.
Assuntos
Caveolinas , Proteínas do Citoesqueleto/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Musculares , Proteínas Oncogênicas de Retroviridae/metabolismo , Transdução de Sinais , Células 3T3 , Aminopeptidases/metabolismo , Animais , Transporte Biológico , Caveolina 1 , Linhagem Celular , Cistinil Aminopeptidase , Transportador de Glucose Tipo 4 , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteína Oncogênica v-cbl , Fosforilação , Técnicas do Sistema de Duplo-HíbridoRESUMO
c-Cbl-associating protein (CAP) is a multifunctional signaling protein that interacts with c-Cbl, facilitating the tyrosine phosphorylation of c-Cbl in response to insulin. In 3T3-L1 adipocytes and diabetic rodents, CAP gene expression is stimulated by activators of peroxisome proliferator activator receptor gamma (PPARgamma), such as thiazolidinediones (TZDs), resulting in increased insulin-stimulated c-Cbl phosphorylation. Sequence analysis of 2.5 kilobases of the 5'-flanking region of the CAP gene reveals a predicted peroxisome proliferator response element (PPRE) from -1085 to -1097. The isolated promoter was functional in 3T3 fibroblasts and adipocytes. Co-transfection of the CAP promoter with PPARgamma and retinoic acid X receptor alpha caused fold stimulation of promoter activity. The TZD rosiglitazone produced an additional 2-3-fold stimulation of the promoter. Deletion of the predicted PPRE from the CAP promoter abolished its ability to respond to rosiglitazone. Gel shift analysis of the putative PPARgamma site demonstrates direct binding of PPAR/retinoid X receptor heterodimers to the PPRE in the CAP gene. These data demonstrate that TZDs directly stimulate transcription of the CAP gene through activation of PPARgamma.
Assuntos
Proteínas do Citoesqueleto/genética , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Células 3T3 , Animais , Sequência de Bases , Clonagem Molecular , DNA , Dimerização , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/metabolismoRESUMO
It is well established that glucocorticoid hormones induce apoptosis in immature developing thymocytes. Thymocyte apoptosis can be modulated by growth factors, anti-oxidants and adhesion receptors. We have previously demonstrated that thymosin alpha1 (Talpha1) antagonizes dexamethasone-induced apoptosis of CD4+CD8+ thymocytes. In the present study, we further characterize the dose and time dependence of Talpha1's antagonism of dexamethasone-induced thymocyte apoptosis. Talpha1 is effective at concentrations ranging from 2 to 100 microg/10(6) thymocytes. Talpha1 pre-treatment is necessary to achieve its anti-apoptotic activity. Talpha1 provides temporary protection to thymocytes by slowing dexamethasone's apoptotic activity up to 12 h post dexamethasone treatment. Additionally, Talpha1's activity is not sensitive to cycloheximide treatment, suggesting Talpha1's activity is independent of protein synthesis. Finally, Talpha1 is unable to antagonize apoptosis induced by the reactive oxygen species, H2O2, suggesting Talpha1's antagonism of dexamethasone occurs at the early stages of dexamethasone-induced apoptosis, prior to the production of reactive oxygen species. This evidence suggests that Talpha1 may provide a mechanism to transiently extend the life of a thymocyte during thymic selection.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anti-Inflamatórios/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Dexametasona/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Timosina/análogos & derivados , Adjuvantes Imunológicos/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Técnicas In Vitro , Masculino , Camundongos , Biossíntese de Proteínas , Linfócitos T/metabolismo , Timalfasina , Timosina/metabolismo , Timosina/farmacologia , Fatores de TempoRESUMO
It is well established that glucocorticoid hormones and anti-CD3 monoclonal antibodies induce apoptosis in immature developing thymocytes. This process can be modulated by soluble factors, anti-oxidants and adhesion receptors. Previously we have demonstrated that thymosin alpha 1 (T alpha 1), a 28-amino acid thymic peptide hormone, is a dose and time dependent antagonist of dexamethasone (DEX) and CD# induced DNA fragmentation of murine thymocytes in vitro. To further investigate the mechanism of T alpha 1 action we determined a T alpha 1 sensitive thymocyte population and examined some of the molecular events associated with T alpha 1 anti-apoptotic activity. Phenotypic analysis of the sub-populations of thymocytes, based on CD4 and CD8 expression, revealed that T alpha 1 exerts its effect on CD4+ CD8+ immature thymocytes. T alpha 1 treatment of thymocytes delays the production of free radicals and the subsequent consumption of glutathione, that is observed during both DEX and CD3 induced apoptosis. We further demonstrate that T alpha 1 stimulates the production of cAMP and activates PKC in thymocytes. These data suggest that T alpha 1 exerts an influence on the development of a population of immature T-cells in the thymus by effecting the sensitivity of thymocytes to apoptosis during the pre-selection stages of thymic development. Our studies also suggest that the mechanism of T alpha 1 action involves the induction of both cAMP and PKC dependent second messenger pathways.
Assuntos
Apoptose , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , AMP Cíclico/metabolismo , Dexametasona/antagonistas & inibidores , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro , Timosina/análogos & derivados , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Radicais Livres , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Timalfasina , Timosina/farmacologia , Timo/citologiaRESUMO
Thymosin fraction 5 (TF5), a thymic preparation, has been shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities. Thymosin alpha1 (T alpha1) was the first biologically active polypeptide to be purified from TF5 and completely characterized. It is an acidic peptide with an isoelectric point of 4.2 and a molecular weight of 3108. T alpha1 is considered a biological response modifier which amplifies T-cell immunity. In the present study, we have studied some pharmacokinetic properties of T alpha1 by measuring its concentrations in serum, urine and ten major organs of female Swiss-Webster mice following administration of 500 microg T alpha1 intraperitoneally. Using a modified enzymatic immunoassay, our data show a significant increase of T alpha1 in serum 2 min after injection and lasting for 2 h (average: 1.55 +/- 0.27 microg/ml). In urine, at four different time points after injection (20 min, 40 min, 2 h, 6 h), increased concentrations of T alpha1 were found between 24.2 and 25.4 microg/ml (average: 25 +/- 0.47 microg/ml). Of the 500 microg T alpha1 administered to mice, 8.97% was recovered at the end of the study, of which 2% corresponded to urine, 1.25% to serum (2 ml of serum per mouse), and 5.72% to organs. Since the urine/day volume and the serum volume of any Swiss Webster mouse is ca 2 ml, additional extrapolation of the above mentioned values could show percentages of recovery close to 40% for urine and 2.5% for serum. In most of the organs, the wet weight concentrations of T alpha1 increased significantly during the first 40 min after injection in comparison to their baseline wet weight concentrations. These organs consisted of the following: thymus (33.1 +/- 3.5 microg/g vs 18 microg/g baseline); lungs (7.7 +/- 1.1 microg/g vs 1.9 microg/g baseline); spleen (15.6 +/- 0.7 microg/g vs 5.6 microg/g); kidneys (6.2 +/- 1.1 microg/g vs 3.9 microg/g); ovaries (9.2 +/- 1.4 microg/g vs 0 microg/g); and peritoneal fat (4 +/- 1 microg/g vs 0 microg/g). No significant increases were observed in the liver (1.7 +/- 0.1 microg/g vs 1.4 microg/g) and heart (0.7 +/- 0.5 microg/g vs 0 microg/g). Increased concentrations of T alpha1 were not detected in the brain and skeletal muscle tissues. These pharmacokinetic studies of T alpha1 in mice indicate that rapid renal excretion of T alpha1 represents a major source of humoral loss following I.P. administration. Recent preliminary studies in humans confirm that the kidney rapidly releases high levels of T alpha1 in urine in a time frame consistent with that observed in mice.
Assuntos
Timosina/análogos & derivados , Tecido Adiposo/metabolismo , Adjuvantes Imunológicos/sangue , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/urina , Animais , Feminino , Humanos , Técnicas Imunoenzimáticas , Rim/metabolismo , Pulmão/metabolismo , Camundongos , Ovário/metabolismo , Baço/metabolismo , Timalfasina , Timosina/sangue , Timosina/farmacocinética , Timosina/urina , Timo/metabolismo , Distribuição TecidualRESUMO
Thymosin beta 4 (T beta 4) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. T beta 4 functions mainly as an actin-sequestering molecule in nonmuscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of T beta 4 in human and other mammals have not been reported so far. In the present study, we have measured T beta 4 concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 micrograms synthetic T beta 4. Using a modified enzymatic immunoassay, our data show a significant increase of T beta 4 in serum starting 2 min after injection and lasting for 40 min (average: 2.34 +/- 0.54 micrograms/ml). High concentrations were found in urine (59.3 +/- 7.54 micrograms/ml) at three different points after injection (20 min, 40 min, and 2 h). Of the 400 micrograms T beta 4 administered to mice 83% was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of T beta 4 increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 micrograms/g), heart (80 micrograms/g), liver (15 micrograms/g vs 9 micrograms/g), kidneys (65 micrograms/g vs 28 micrograms/g), and peritoneal fat (47 micrograms/g vs 13 micrograms/g). Wet weight concentrations increased in the thymus (196 micrograms/g vs 147 micrograms/g) and muscle (45 micrograms/g vs 0 micrograms/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 micrograms/g vs 161 micrograms/g). Ovaries had a biphasic increase at 40 min (72 micrograms/g vs 62 micrograms/g) and 24 h (92 micrograms/g vs 62 micrograms/g) after T beta 4 administration. In lungs, the highest wet weight increase after injection (149 micrograms/g at timepoint 6 h) was not higher than its basal wet weight concentration (153 micrograms/g). These pharmacokinetic studies of T beta 4 in mice have established that high levels of T beta 4 are found in blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of T beta 4 during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.
Assuntos
Camundongos/metabolismo , Timosina/farmacocinética , Animais , Feminino , Humanos , Técnicas Imunoenzimáticas , Injeções Intraperitoneais , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Timosina/sangue , Timosina/urina , Distribuição TecidualRESUMO
Thirty normocholesterolemic laying hens were used to investigate the effect of oral administration of Eubacterium coprostanoligenes on plasma cholesterol concentrations. Hens were divided randomly into three treatment groups (active, inactive, and control) with 10 hens in each group. The active group received 0.5 mL of E. coprostanoligenes suspension (approximately 2 x 10(7) cells per milliliter) daily for 4 wk; the inactive group received the same dosage of killed (boiled) bacterial suspension; and the control group received no supplemental bacteria. After bacterial feeding, the coprostanol to cholesterol ratio in feces of the active group was significantly greater than ratios of the inactive and control groups, indicating that E. coprostanoligenes was colonized in the intestine of hens and was converting intestinal cholesterol to coprostanol. Plasma cholesterol concentrations, however, were not affected by the bacterial treatment.