Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.
(33/18461)
Protein tyrosine phosphatase-1B (PTP-1B) has been implicated in the negative regulation of insulin signaling. Disruption of the mouse homolog of the gene encoding PTP-1B yielded healthy mice that, in the fed state, had blood glucose concentrations that were slightly lower and concentrations of circulating insulin that were one-half those of their PTP-1B+/+ littermates. The enhanced insulin sensitivity of the PTP-1B-/- mice was also evident in glucose and insulin tolerance tests. The PTP-1B-/- mice showed increased phosphorylation of the insulin receptor in liver and muscle tissue after insulin injection in comparison to PTP-1B+/+ mice. On a high-fat diet, the PTP-1B-/- and PTP-1B+/- mice were resistant to weight gain and remained insulin sensitive, whereas the PTP-1B+/+ mice rapidly gained weight and became insulin resistant. These results demonstrate that PTP-1B has a major role in modulating both insulin sensitivity and fuel metabolism, thereby establishing it as a potential therapeutic target in the treatment of type 2 diabetes and obesity. (+info)
AP-4, a novel protein complex related to clathrin adaptors.
(34/18461)
Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells. (+info)
Modifications to rat lens major intrinsic protein in selenite-induced cataract.
(35/18461)
PURPOSE: To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m-calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS: Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS: The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239, corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235. CONCLUSIONS: Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei. (+info)
Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification.
(36/18461)
Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis. (+info)
Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family.
(37/18461)
Hoxa2 is expressed in cranial neural crest cells that migrate into the second branchial arch and is essential for proper patterning of neural-crest-derived structures in this region. We have used transgenic analysis to begin to address the regulatory mechanisms which underlie neural-crest-specific expression of Hoxa2. By performing a deletion analysis on an enhancer from the Hoxa2 gene that is capable of mediating expression in neural crest cells in a manner similar to the endogenous gene, we demonstrated that multiple cis-acting elements are required for neural-crest-specific activity. One of these elements consists of a sequence that binds to the three transcription factor AP-2 family members. Mutation or deletion of this site in the Hoxa2 enhancer abrogates reporter expression in cranial neural crest cells but not in the hindbrain. In both cell culture co-transfection assays and transgenic embryos AP-2 family members are able to trans-activate reporter expression, showing that this enhancer functions as an AP-2-responsive element in vivo. Reporter expression is not abolished in an AP-2(alpha) null mutant embryos, suggesting redundancy with other AP-2 family members for activation of the Hoxa2 enhancer. Other cis-elements identified in this study critical for neural-crest-specific expression include an element that influences levels of expression and a conserved sequence, which when multimerized directs expression in a broad subset of neural crest cells. These elements work together to co-ordinate and restrict neural crest expression to the second branchial arch and more posterior regions. Our findings have identified the cis-components that allow Hoxa2 to be regulated independently in rhombomeres and cranial neural crest cells. (+info)
Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.
(38/18461)
We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals. (+info)
Missense mutations in the gp91-phox gene encoding cytochrome b558 in patients with cytochrome b positive and negative X-linked chronic granulomatous disease.
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Chronic granulomatous disease (CGD) is a disorder of host defense due to genetic defects of the superoxide (O2-) generating NADPH oxidase in phagocytes. A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the oxidase. The X-linked form of the disease is due to defects in the gp91-phox gene. We report here biochemical and genetic analyses of patients with typical and atypical X-linked CGD. Immunoblots showed that neutrophils from one patient had small amounts of p22-phox and gp91-phox and a low level of O2- forming oxidase activity, in contrast to the complete absence of both subunits in two patients with typical CGD. Using polymerase chain reactions (PCR) on cDNA and genomic DNA, we found novel missense mutations of gp91-phox in the two typical patients and a point mutation in the variant CGD, a characteristic common to two other patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient provided evidence for the presence of heme of cytochrome b558. Recently, we reported another variant CGD with similar amounts of both subunits, but without oxidase activity or the heme spectrum. A predicted mutation at amino acid 101 in gp91-phox was also confirmed in this variant CGD by PCR of the genomic DNA. These results on four patients, including those with two variant CGD, are discussed with respect to the missense mutated sites and the heme binding ligands in gp91-phox. (+info)
The multisubstrate docking site of the MET receptor is dispensable for MET-mediated RAS signaling and cell scattering.
(40/18461)
The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association-kinase assay we found that the mutated receptor can still associate and phosphorylate a approximately 250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering. (+info)