RESUMEN
Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mitógenos/farmacología , Mutación , Animales , Células CHO , Dominio Catalítico/genética , Cricetinae , Dimerización , Proteína Adaptadora GRB2 , Transferasas Intramoleculares/metabolismo , Mitosis , Fosforilación , Transporte de Proteínas , Proteínas/metabolismo , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Rubidio/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.
Asunto(s)
Glucosa/metabolismo , Resistencia a la Insulina/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/fisiología , Adipocitos/fisiología , Tejido Adiposo Pardo , Animales , Transporte Biológico , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Glucógeno/metabolismo , Glucólisis , Heterocigoto , Hígado/metabolismo , Ratones , Ratones Noqueados , Proteínas Qa-SNARERESUMEN
To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.
Asunto(s)
Insulina/metabolismo , Proteínas de la Membrana/genética , Proteínas Musculares , Condicionamiento Físico Animal , Proteínas de Transporte Vesicular , Adipocitos/metabolismo , Animales , Glucemia/metabolismo , Western Blotting , Peso Corporal/genética , Bovinos , Células Cultivadas , ADN Complementario/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/metabolismo , Eliminación de Gen , Glucosa/farmacocinética , Transportador de Glucosa de Tipo 4 , Homocigoto , Hipoxia , Insulina/sangre , Masculino , Proteínas de la Membrana/química , Ratones , Modelos Genéticos , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/metabolismo , Mutagénesis Sitio-Dirigida , Fagocitosis , Pinocitosis , Isoformas de Proteínas , Receptores de Transferrina/metabolismo , Proteínas SNARE , Factores Sexuales , Factores de Tiempo , Distribución Tisular , Transferrina/metabolismo , Proteína 3 de Membrana Asociada a VesículasRESUMEN
Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Insulina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Adipocitos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Línea Celular , Clonación Molecular , Cricetinae , Genes Dominantes , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Orgánulos/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Levaduras/genéticaAsunto(s)
Insulina/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Compartimento Celular , Endocitosis , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas SNARE , Vesículas Sinápticas/metabolismoRESUMEN
Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up-regulation of translation of mRNAs with polypyrimidine tracts at their 5' start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within < 15 min) following rehydration and coincides with the onset of ribosomal S6 subunit phosphorylation. This S6 kinase activity displays chromatographic and biochemical characteristics that are similar to those of mammalian p70S6k. Partially purified Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.
Asunto(s)
Artemia/embriología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía Liquida/métodos , Citosol/enzimología , Deshidratación , Embrión no Mamífero/enzimología , Embrión no Mamífero/fisiología , Activación Enzimática/efectos de los fármacos , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fosfoproteínas Fosfatasas/farmacología , Proteína Fosfatasa 2 , Proteínas Quinasas S6 Ribosómicas/efectos de los fármacos , Especificidad por SustratoRESUMEN
The epidermal growth factor (EGF) receptor, like other protein tyrosine kinases, shows a preference for substrates having acidic residues in the vicinity of the tyrosyl residue that undergoes phosphorylation. We have developed a peptide substrate for the EGF receptor, termed tyrsub, which is based upon the highly acidic amino terminal sequence of human erythrocyte Band 3. Tyrsub possesses the lowest apparent Km(Km(app) = 32 microM) for phosphorylation by the EGF receptor of any peptide substrate reported to date. Using tyrsub, as well as analogs containing either Ser (sersub) or Phe (phesub) in place of Tyr, we investigated the relative importance of characteristics of the hydroxyaminoacyl residue in substrate recognition. Sersub was unable either to act as a substrate or serve as an effective inhibitor of tyrsub phosphorylation by the EGF receptor. Phesub was also unable to inhibit EGF-stimulable tyrsub phosphorylation, suggesting that the phenolic hydroxyl of the tyrosyl residue, rather than the aromatic ring, predominates in substrate recognition. These results indicate that for peptide substrates, at least, binding consists of two steps, recognition, in which the tyrosyl side chain plays the central role, and docking, in which residues surrounding the tyrosyl residue contribute to stabilizing binding interactions.
Asunto(s)
Receptores ErbB/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Receptores ErbB/antagonistas & inhibidores , Gastrinas/farmacología , Cinética , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Fosforilación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The residue proposed to serve as the catalytic base for phosphoryl transfer, Asp-813, of the human epidermal growth factor receptor (EGFR) was mutated to Ala, and the mutant receptor (D813A) was expressed in Chinese hamster ovary (CHO) cells. Partially purified D813A exhibited no detectable kinase activity in the absence or presence of EGF. A low level of EGF-stimulable phosphorylation of D813A was detectable in intact cells, apparently due to the activity of an associated Tyr kinase(s). As previously observed for kinase-inactive Lys-721 mutants, EGF binding to D813A stimulates mitogen-activated protein kinase activity. Surprisingly, and unlike results reported for Lys-721 mutants, D813A is capable of stimulating both 86Rb+ uptake and DNA synthesis in response to EGF. These data suggest not only that Asp-813 is critical to the catalytic activity of the EGFR but also that differences may exist in the signaling properties of kinase-negative Lys-721 and kinase-negative Asp-813 EGFR mutants.