Biomedical subjects

S Shoelson

Publications and source records attributed to S Shoelson.

12 recordsLinked to original sources

PubMed1997

Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes.

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based motif in their cytosolic domains by an adaptor (AP-2) complex. The mu2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based motif. AP-2 is the first protein known (in any context) to interact with phosphatidylinositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.

Adaptor Protein Complex 1↗

PubMed1996

Functional roles of the Shc phosphotyrosine binding and Src homology 2 domains in insulin and epidermal growth factor signaling.

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

Animals↗

PubMed1996

p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase.

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

Adaptor Proteins, Signal Transducing↗

PubMed1995

A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.

BACKGROUND: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. RESULTS: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. CONCLUSION: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.

3T3 Cells↗

PubMed1994

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

Amino Acid Sequence↗

PubMed1993

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.

Amino Acid Sequence↗

PubMed1993

Characterization of insulin autoantibodies in relatives of patients with type I diabetes.

We studied in detail the anti-insulin autoantibodies in 29 nondiabetic relatives of patients with type I diabetes. The affinity of the autoantibodies for [125I]human insulin was high (1.34 x 10(9)-20.71 x 10(9) L/mol), and the capacity was low (0.84 x 10(-12)-37.80 x 10(-12) M). The product of affinity x capacity of each relative's antibodies directly correlated (r = 0.99) with the level of antibodies determined in our standard radioassay. The autoantibodies from each of the subjects studied had the same rank order of affinities for insulin from different species. Guinea pig, fish insulin, and insulin containing Trp rather than Leu in position 13 of the A-chain inhibited minimally the human insulin binding. Human proinsulin, insulin containing Gln rather than Glu in position 17 of the A-chain, and desoctapeptide insulin (des B23-30) all inhibited binding effectively. Insulin autoantibodies in relatives of patients with type I diabetes share common epitope(s), which suggests a common pathogenic mechanism for production of such antibodies. The epitopes from this initial analysis appear to include amino acids B1-B3 and A8-A13. The region recognized can be distinguished from the insulin receptor binding domain.

Adolescent↗

PubMed1993

Identification of residues in the beta platelet-derived growth factor receptor that confer specificity for binding to phospholipase C-gamma 1.

The SH2 domains of cytoplasmic signaling proteins bind to autophosphorylated growth factor receptors by direct recognition of specific phosphotyrosine-containing sites. To identify the phosphotyrosine involved in association of phospholipase C (PLC)-gamma 1 with the beta platelet-derived growth factor receptor (PDGFR), and to investigate which contiguous residues confer specificity for PLC-gamma 1, phosphotyrosine-containing glutathione S-transferase (GST) fusion proteins possessing different regions of the beta-PDGFR were incubated with lysates of Rat-2 cells that overexpress PLC-gamma 1. The phosphorylated C-terminal tail of the PDGFR bound PLC-gamma 1, but did not associate with phosphatidylinositol (PI) 3'-kinase or GTPase-activating protein (GAP). High-affinity binding of PLC-gamma 1 was dependent on phosphorylation of Tyr-1021. Creation of a new phosphorylation site by replacing Asp-1018 with tyrosine did not restore binding of PLC-gamma 1 in the absence of Tyr-1021, indicating that the location of the phosphorylated tyrosine is important for PLC-gamma 1 binding. Substitution of the proline at the +3 position relative to Tyr-1021 with methionine (Y1021IIP-->Y1021IIM) in the phosphorylated PDGFR tail did not alter PLC-gamma 1 association, but conferred binding activity towards PI 3'-kinase, indicating that this residue is critical in discriminating between PLC-gamma 1 and PI 3'-kinase. Progressive conversion of the three residues C-terminal to Tyr-1021 to the consensus for PI 3'-kinase binding (YMDM) allowed PI 3'-kinase association, but did not block PLC-gamma 1 binding, suggesting that additional residues other than the three residues immediately following the phosphotyrosine may contribute to the association of PLC-gamma 1 with the PDGFR. These results indicate that phosphorylation at Tyr-1021 in the tail of the PDGFR creates a specific binding site for PLC-gamma 1. Proline at the +3 position relative to Tyr-1021 is crucial in conferring specificity for binding to PLC-gamma 1.

Amino Acid Sequence↗

PubMed1992

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.

3T3 Cells↗

PubMed1983

Three mutant insulins in man.

We have previously identified a structurally abnormal insulin in the serum and pancreas of a middle-aged man with diabetes mellitus which arose from a leucine for phenylalanine substitution at position 24 or 25 of the insulin B chain; further analysis of the patient's leukocyte DNA showed that one of the patient's insulin alleles had undergone mutation resulting in loss of an MboII restriction site normally present in the human insulin gene. Two additional and unrelated patients with the same clinical syndrome have now been identified (ref. 4 and unpublished results). All of these patients showed hyperglycaemia typical of diabetes and with marked hyperinsulinaemia typical of insulin resistance, but all three show normal tolerance to exogenously administered insulin. As the opportunity of examining pancreatic tissue from patients suspected of secreting insulin variants is rare, we have developed a method combining HPLC and radioimmunoassay to identify insulin variants isolated from human sera. By this method we have shown that all three patients noted above secrete structurally variant and chemically distinct insulins. In correction of our original assignment, one is identified as [LeuB25]insulin.

Amino Acid Sequence↗

PubMed1983

Identification of a mutant human insulin predicted to contain a serine-for-phenylalanine substitution.

Using information gained from (i) the relative HPLC retention of an abnormal insulin present in the serum of a hyperinsulinemic diabetic patient and (ii) the loss of an Mbo II restriction site in one of the patient's insulin gene alleles, it was recently predicted that the mutant insulin contained a serine-for-phenylalanine substitution at position B24 or B25. We have now prepared human [SerB24]insulin and [SerB25]insulin by solid-phase peptide synthesis and semisynthesis using an improved approach whereby the protecting groups can be removed from the final product in a single step. During reversed-phase HPLC analysis, the two semisynthetic insulins were clearly separated from normal insulin and from each other. Analysis of the patient's immunoaffinity-purified serum insulin by HPLC and radioimmunoassay showed that the insulin eluted at the position of [SerB24]insulin and coeluted with the analog when the two were studied in admixture. Additional studies showed that [SerB24]insulin and [SerB25]insulin have about 16% and 0.5% of the activity of normal insulin, respectively, in stimulating glucose oxidation by isolated rat adipocytes. We conclude that the patient's abnormal insulin (insulin Los Angeles) is human [SerB24]insulin and that abnormal insulins with amino acid replacements at both positions B24 and B25 can be associated with human diabetes.

Amino Acid Sequence↗

PubMed1982

Evidence for the presence of somatostatin 28 in plasma.

Somatostatin-like immunoreactivity (SLI) from dog and rat plasma eluted from Biogel P-6 columns as three distinct peaks. A large-molecular-weight peak was present in the void volume of the column, an intermediate-sized peak (SLI28) coeluted with synthetic somatostatin 28 (S-28), and a small-molecular-weight peak (SLI14) coeluted with SRIF. Material from the SLI28 peak diluted in parallel to the S-28 standard in the radioimmunoassay and behaved identically to S-28 on high pressure liquid chromatography (HPLC). Levels of SLI28 in the portal vein were consistently greater than the simultaneously measured peripheral levels (portal peripheral ratio 2.2 +/- 0.2). Venous samples drawn from multiple sites suggested that SLI28 is segregated by the duodenum and/or pancreas and the intestine. This data is consistent with the possibility that S-28 is a hormone distinct from SRIF.

Animals↗