Biomedical subjects

S E Shoelson

Publications and source records attributed to S E Shoelson.

At least 19 recordsLinked to original sources

PubMed2003

Identification and functional characterization of a novel mutation of hepatocyte nuclear factor-1alpha gene in a Korean family with MODY3.

AIMS/HYPOTHESIS: After screening 16 Korean families with early onset Type 2 diabetes in search for hepatocyte nuclear factor (HNF) -1alpha gene mutation, we identified a novel missense mutation (R263L) associated with MODY phenotype. We studied the biological characteristics of the mutation and the potential functional consequences based on the crystallographic structure of HNF-1alpha in complex with DNA. METHODS: DNA from subjects with a familial form of early onset diabetes was isolated and HNF-1alpha was sequenced. The R263L substitution was generated by PCR-based sited-directed mutagenesis. Functional and biochemical studies were conducted by reporter assay using glucose-transporter type 2 (GLUT2) or insulin promoters and electrophoretic mobility shift assay, respectively. RESULTS: Transfection of wild-type HNF-1alpha increased the reporter activities of GLUT2 and insulin promoters in NIH3T3 and SK-Hep1 cells, while R263L mutant was defective in transactivation of those promoters. Both wild-type HNF-1alpha and R263L mutant could not transactivate GLUT2 and insulin promoters in MIN6N8 insulinoma cells. R263L mutant had a defective cooperation with its heterodimeric partner HNF-1beta or coactivator p300. R263L mutant protein displayed greatly reduced DNA binding ability, despite its comparable protein stability to the wild-type HNF-1alpha. CONCLUSION/INTERPRETATION: These results suggest that the mutation of HNF-1alpha at codon 263 from arginine to leucine leads to the development of MODY3 through decreased insulin production and defective glucose sensing. These findings are in good agreement with the crystal structure in which R263 makes hydrogen bonds with phosphorus atoms of DNA backbone to mediate the stable binding of HNF-1alpha homeodomain to the promoter.

Amino Acid Substitution↗

PubMed2003

Inflammation and the IKK beta/I kappa B/NF-kappa B axis in obesity- and diet-induced insulin resistance.

Antidiabetic effects associated with salicylates have been known for years, although the underlying mechanisms were not understood. We have been reinvestigating these effects in the light of recent discoveries in the areas of signal transduction and insulin resistance. Our findings showed that signaling pathways leading to I kappa B kinase beta (IKK beta) and NF-kappa B are activated in insulin-responsive tissues of obese and high-fat-fed animals. Since activation correlates with the development of insulin resistance, we asked whether signaling through this might be involved in the pathogenesis of insulin resistance. Heterozygous gene deletion (Ikk beta+/-) or salicylates, working as IKK beta inhibitors, improved insulin sensitivity in insulin-resistant rodent models. Furthermore, high doses of salicylates (aspirin or salicylate) improved insulin sensitivity in patients with type II diabetes. Our studies implicate an inflammatory process in the pathogenesis of insulin resistance in obesity and type II diabetes mellitus and identify the IKK beta/NF-kappa B pathway as a molecular mediator of insulin resistance and pharmacological target for insulin sensitization.

Animals↗

PubMed2001

Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta.

We show that high doses of salicylates reverse hyperglycemia, hyperinsulinemia, and dyslipidemia in obese rodents by sensitizing insulin signaling. Activation or overexpression of the IkappaB kinase beta (IKKbeta) attenuated insulin signaling in cultured cells, whereas IKKbeta inhibition reversed insulin resistance. Thus, IKKbeta, rather than the cyclooxygenases, appears to be the relevant molecular target. Heterozygous deletion (Ikkbeta+/-) protected against the development of insulin resistance during high-fat feeding and in obese Lep(ob/ob) mice. These findings implicate an inflammatory process in the pathogenesis of insulin resistance in obesity and type 2 diabetes mellitus and identify the IKKbeta pathway as a target for insulin sensitization.

Animals↗

PubMed2001

Prevention of fat-induced insulin resistance by salicylate.

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and may involve fat-induced activation of a serine kinase cascade involving IKK-beta. To test this hypothesis, we first examined insulin action and signaling in awake rats during hyperinsulinemic-euglycemic clamps after a lipid infusion with or without pretreatment with salicylate, a known inhibitor of IKK-beta. Whole-body glucose uptake and metabolism were estimated using [3-(3)H]glucose infusion, and glucose uptake in individual tissues was estimated using [1-(14)C]2-deoxyglucose injection during the clamp. Here we show that lipid infusion decreased insulin-stimulated glucose uptake and activation of IRS-1-associated PI 3-kinase in skeletal muscle but that salicylate pretreatment prevented these lipid-induced effects. To examine the mechanism of salicylate action, we studied the effects of lipid infusion on insulin action and signaling during the clamp in awake mice lacking IKK-beta. Unlike the response in wild-type mice, IKK-beta knockout mice did not exhibit altered skeletal muscle insulin signaling and action following lipid infusion. In summary, high-dose salicylate and inactivation of IKK-beta prevent fat-induced insulin resistance in skeletal muscle by blocking fat-induced defects in insulin signaling and action and represent a potentially novel class of therapeutic agents for type 2 diabetes.

Animals↗

PubMed2000

Shf, a Shb-like adapter protein, is involved in PDGF-alpha-receptor regulation of apoptosis.

Recent work has implicated the importance of adapter proteins in signal transduction. To identify homologues of the previously identified adapter protein Shb, database searches were performed. A Shb-like protein was found which we have named Shf. Shf contains an SH2 domain and four putative tyrosine phosphorylation sites and is mainly expressed in skeletal muscle, brain, liver, prostate, testis, ovary, small intestine, and colon. The SH2 domain of Shf bound to the PDGF-alpha-receptor at tyrosine-720, but not to the PDGF-beta-receptor in PAE cells. Pervanadate induced tyrosine phosphorylation of Shf in NIH3T3 fibroblasts overexpressing this protein, whereas PDGF-AA alone had no detectable effect. NIH3T3 cells overexpressing Shf displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. Our findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.

3T3 Cells↗

PubMed2000

Crystal structure of human frataxin.

Friedreich's ataxia, an autosomal recessive neurodegenerative disorder characterized by progressive gait and limb ataxia, cardiomyopathy, and diabetes mellitus, is caused by decreased frataxin production or function. The structure of human frataxin, which we have determined at 1.8-A resolution, reveals a novel protein fold. A five-stranded, antiparallel beta sheet provides a flat platform, which supports a pair of parallel alpha helices, to form a compact alphabeta sandwich. A cluster of 12 acidic residues from the first helix and the first strand of the large sheet form a contiguous anionic surface on the protein. The overall protein structure and the anionic patch are conserved in eukaryotes, including animals, plants, and yeast, and in prokaryotes. Additional conserved residues create an extended 1008-A(2) patch on a distinct surface of the protein. Side chains of disease-associated mutations either contribute to the anionic patch, help create the second conserved surface, or point toward frataxin's hydrophobic core. These structural findings predict potential modes of protein-protein and protein-iron binding.

Amino Acid Sequence↗

PubMed2000

Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps.

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.

Animals↗

PubMed1999

Crystal structure of the pleckstrin homology-phosphotyrosine binding (PH-PTB) targeting region of insulin receptor substrate 1.

We have determined the crystal structure at 2.3-A resolution of an amino-terminal segment of human insulin receptor substrate 1 that encompasses its pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Both domains adopt the canonical seven-stranded beta-sandwich PH domain fold. The domains are closely associated, with a 720-A(2) contact surface buried between them that appears to be stabilized by ionic, hydrophobic, and hydrogen bonding interactions. The nonconserved 46-residue linker between the domains is disordered. The PTB domain peptide binding site is fully exposed on the molecular surface, as is a large cationic patch at the base of the PH domain that is a likely binding site for the head groups of phosphatidylinositol phosphates. Binding assays confirm that phosphatidylinositol phosphates bind the PH domain, but not the PTB domain. Ligand binding to the PH domain does not alter PTB domain interactions, and vice versa. The structural and accompanying functional data illustrate how the two binding domains might act cooperatively to effectively increase local insulin receptor substrate 1 concentration at the membrane and transiently fix the receptor and substrate, to allow multiple phosphorylation reactions to occur during each union.

Amino Acid Sequence↗

PubMed1998

Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site.

In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXXphi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated by phosphoinositide lipids that are phosphorylated at the D-3 position of the inositol ring, and is also enhanced by the formation of clathrin-AP-2 coats. Here, we describe the detection of a specific interaction between peptides containing a second sorting motif, the dileucine motif, and AP-1, the clathrin adaptor complex responsible for sorting proteins at the trans-Golgi network (TGN). Surprisingly, the site of dileucine binding is the beta1 subunit, not mu 1. A YXXphi-containing peptide from a protein trafficked within the TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphate and 3,4, 5-triphosphate did not activate the interaction between dileucine-containing peptides and AP-1 but instead inhibited it, and clathrin-AP-1 coat formation did not alter the interaction. Thus, there are at least two physically separate binding sites for sorting signals on APs, which are also regulated independently.

Amino Acid Sequence↗

PubMed1998

Crystal structure of the tyrosine phosphatase SHP-2.

The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains. In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site. This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft. Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface. Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme. Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation.

Crystallography↗

PubMed1998

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins.

Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Tandem SH2 domains confer high specificity in tyrosine kinase signaling.

SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-gamma1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the approximately 20-50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.

Amino Acid Sequence↗

PubMed1998

Identification of SOCS-3 as a potential mediator of central leptin resistance.

Leptin affects food intake and body weight by actions on the hypothalamus. Although leptin resistance is common in obesity, mechanisms have not been identified. We examined the effect of leptin on expression of the suppressors-of-cytokine-signaling (SOCS) family of proteins. Peripheral leptin administration to ob/ob, but not db/db mice, rapidly induced SOCS-3 mRNA in hypothalamus, but had no effect on CIS, SOCS-1, or SOCS-2. A leptin-dependent increase of SOCS-3 mRNA was seen in areas of hypothalamus expressing high levels of the leptin receptor long form. In mammalian cell lines, SOCS-3, but not CIS or SOCS-2, blocked leptin-induced signal transduction. Expression of SOCS-3 mRNA in the arcuate and dorsomedial hypothalamic nuclei is increased in Ay/a mice, a model of leptin-resistant murine obesity. In conclusion, SOCS-3 is a leptin-inducible inhibitor of leptin signaling, and a potential mediator of leptin resistance in obesity.

Animals↗

PubMed1998

Correlation between binding and dynamics at SH2 domain interfaces.

Protein recognition is a key determinant in regulating biological processes. Structures of complexes of interacting proteins provide significant insights into the mechanism of specific recognition. However, studies performed by modifying residues within a protein interface demonstrate that binding is not fully explained by these static pictures. Thus, structural data alone was not predictive of affinities in binding studies of phospholipase Cgamma1 and Syp phosphatase SH2 domains with phosphopeptides. NMR relaxation experiments probing dynamics of methyl groups of these complexes indicate a correlation between binding energy and restriction of motion at the interfacial region responsible for specific binding.

Insulin Receptor Substrate Proteins↗

PubMed1997

The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70.

The Cbl protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cbl physically interacts with tyrosine kinases is of substantial interest. We recently identified a phosphotyrosine binding (PTB) domain residing within the N-terminal transforming region of Cbl (Cbl-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the Cbl-PTB domain selects a D(N/D)XpY motif, reminiscent of but distinct from the NPXpY motif recognized by the PTB domains of Shc and IRS-1/2. A phosphopeptide predicted by this motif and corresponding to the in vivo negative regulatory phosphorylation site of ZAP-70 (Tyr(P)292) specifically inhibited binding of ZAP-70 to Cbl-N. A ZAP-70/Y292F mutant failed to bind to Cbl-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cbl-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cbl-N or full-length Cbl in vivo. These results identify a potential Cbl-PTB domain-dependent role for Cbl in the negative regulation of ZAP-70 and predict potential Cbl-PTB domain binding sites on other protein tyrosine kinases known to interact with Cbl.

3T3 Cells↗

PubMed1997

Conformational changes of the insulin receptor upon insulin binding and activation as monitored by fluorescence spectroscopy.

We have characterized the changes in intrinsic fluorescence that the insulin receptor undergoes upon ligand binding and autophosphorylation. The binding of insulin to its receptor results in an increase in the receptor's fluorescence intensity, emission energy and anisotropy. We monitored the time course of the anisotropy change, and these data, coupled with studies monitoring the energy transfer from insulin receptor tryptophan donors to a fluorescent-labeled insulin, allowed us to conclude that the change in anisotropy is due to a conformational change in the receptor induced by hormone binding. Since insulin association is very fast, the time course also allowed us to estimate the slower rate of formation of this conformationally-altered state. The time course of receptor autophosphorylation was measured under similar conditions and was found to be similar to the ligand-induced anisotropy time course. The simultaneous use of two fluorescent-labeled insulin analogs also allowed us to assess the maximum distance between the two hormones bound to the receptor. Addition of ATP produces a large, seemingly instantaneous increase in anisotropy. Our observation that ATP binds to the insulin receptor in the presence and absence of insulin supports the idea that the conformational change produced by insulin binding increases the rate of autophosphorylation rather than increases ATP affinity. A suggested model for these changes is presented.

3T3 Cells↗

PubMed1997

Human GRB-IRbeta/GRB10. Splice variants of an insulin and growth factor receptor-binding protein with PH and SH2 domains.

cDNA clones encoding human (h) Grb7 and a previously unknown protein with high homology to hGrb-IR and mGrb10 (where m indicates mouse) were found by screening expressed sequence tag data bases. hGrb7 mRNA expression is greatest in pancreas and restricted to a few other tissues. The second protein termed hGrb-IRbeta/Grb10 contains an intact PH domain and lacks the 80-residue mGrb10 insertion. Expression is greatest in pancreas and muscle but occurs in nearly all tissues. hGrb-IRbeta/Grb10 and hGrb-IR likely arise as alternative mRNA splicing products of a common gene. Reverse transcriptase-coupled polymerase chain reaction shows both mRNAs in muscle. In cells, Grb-IRbeta/Grb10 protein translocates from cytosol to membrane upon insulin stimulation, most likely due to direct interactions with the insulin receptor. These interactions are mediated by the SH2 domain and additional regions of the protein. Studies with mutated receptors and synthetic phosphopeptides show that the hGrb-IRbeta/Grb10 SH2 domain binds at least two sites in the insulin receptor: the kinase activation loop > the juxtamembrane site. hGrb-IRbeta/Grb10 also binds a 135-kDa phosphoprotein in unstimulated 3T3-L1 adipocytes; binding is reduced upon insulin stimulation. In addition, the c-Abl SH3 domain binds Grb-IR/Grb10, whereas Fyn, phosphatidylinositol 3-kinase p85, and Grb2 SH3 domains do not. The site of c-Abl SH3 domain interaction is highly conserved within the Grb-IR/Grb10/Grb7/Grb14 family. hGrb-IRbeta/Grb10 also binds platelet-derived growth factor and epidermal growth factor receptors, suggesting a broader role in the signaling pathways of numerous receptors. We conclude that hGrb-IRbeta/Grb10 is a widely expressed, PH and SH2 domain-containing, SH3 domain-binding protein that functions downstream from activated insulin and growth factor receptors.

3T3 Cells↗

PubMed1997

The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling.

We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-gamma1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle.

Adaptor Proteins, Signal Transducing↗