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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP.
Mol Cell Biol 1996 Nov
PMID:The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein. 888 53

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor beta-subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vandate-induced mitogenesis is associated with the selective induction of c-jun and junB expression without significantly inducing c-fos or c-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation and c-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation and c-jun and junB expression may serve the critical roles in mediating vandate-induced mitogenesis in SV40-transformed cells.
Mol Cell Biochem
PMID:Unique and selective mitogenic effects of vanadate on SV40-transformed cells. 892 49

Insulin is a key hormone regulating glucose homeostasis and has many cellular effects on metabolism, growth, and differentiation. Insulin action is mediated through a specific cell-surface receptor. The first step following insulin binding consists in receptor autophosphorylation and stimulation of its tyrosine kinase activity. Among the multiple substrates, the insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate for insulin. IRS-1 binds several Src homology 2 (SH2) proteins through its multiple tyrosine phosphorylation sites: phosphatidylinositol 3-kinase (PI 3-kinase), the Ras guanine-nucleotide-releasing complex Grb2-SOS, the tyrosine phosphatase Syp, and the adapter protein Nck. IRS-1 is essential for many, but not all of the insulin's biological responses. Recently, a primary alternative substrate, i.e. IRS-2, was purified and cloned. Numerous biochemical abnormalities of the insulin signaling system lead to insulin resistance. No doubt, the recent data about the molecular mechanisms of insulin action will provide new insights into the pathophysiology and therapy of diabetes and other insulin resistant states.
Mol Med (Sofia) 1996
PMID:[The insulin transduction system]. 898 14

Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.
Mol Endocrinol 1997 Jan
PMID:Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport. 899 89

Signal transduction by insulin and IGF-1, several interleukins (IL-2, IL-4, IL-9, IL-13), interferons, GH, and other cytokines involves IRS proteins, which link the receptors for these factors to signaling molecules with Src homology-2 domains (SH2-proteins). We recently reported the amino acid sequence of murine IRS-2; in order to examine a potential genetic role for this molecule in disease, we isolated the murine IRS-2 gene and compared the expression pattern of IRS-2 against IRS-1. Like IRS-1, IRS-2 is encoded by a single exon. Whereas IRS-1 is located on murine chromosome 1, IRS-2 is located on murine chromosome 8 near the insulin receptor. IRS-2 is expressed together with IRS-1 in many cells and tissues; however, IRS-2 predominates in murine hematopoietic cells where it may be essential for cytokine signaling; IRS-1 predominates in adipocytes and differentiated 3T3-L1 cells where it contributes to the normal insulin response. In 32D cells, IRS-1 and IRS-2 undergo differential tyrosine phosphorylation during insulin or IL-4 stimulation, as assessed indirectly by interaction with various recombinant SH2 domains. Thus, signaling specificity through the IRS proteins may be accomplished by specific expression patterns and distinct phosphorylation patterns during interaction with various activated receptors.
Mol Endocrinol 1997 Feb
PMID:The IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter for insulin and cytokine action. 901 72

Shc and insulin receptor substrate 1 (IRS-1) are cytoplasmic substrates of tyrosine kinase receptors that engage, localize, and activate downstream SH2 enzymes. Each contains a phosphotyrosine-binding (PTB) domain that is structurally unrelated to SH2 domains. We have designed high-affinity, cellular inhibitors of the Shc PTB domain by incorporating nonnatural, phosphatase-resistant amino acids into short peptides. None of the inhibitors bind the IRS-1 PTB domain, consistent with distinct specificities for domains. The best inhibitor of the Shc domain was introduced by electroporation into Rat1 fibroblasts that express human insulin receptors. Insulin-stimulated phosphorylation of Shc was inhibited, with no effect on IRS-1, and downstream effects on mitogen-activated protein kinase and DNA synthesis were both inhibited. The PTB domain inhibitor had less influence on epidermal growth factor-induced effects and essentially no impact on serum- or phorbol ester-induced effects. The inhibitor did not affect insulin internalization and its degradation. We conclude that the PTB domain of Shc is critical for its phosphorylation by the insulin receptor, that Shc is an important mediator of insulin's mitogenic effects, and that Shc is not central to insulin receptor cycling in these cells. PTB domains can be inhibited selectively in cells and represent potential targets for drug discovery.
Mol Cell Biol 1997 Mar
PMID:Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking. 903 45

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.
Mol Cell Biol 1997 Mar
PMID:Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells. 903 79

Insulin acts on its target tissues by specific interaction with the cell surface insulin receptor (IR). The IR possesses an intrinsic tyrosine kinase (TK) activity which is stimulated by insulin binding. This TK activity is required for many aspects of insulin signalling. We had earlier reported that human plasma alpha 2-HS glycoprotein (alpha 2-HSG) inhibits insulin-stimulated mitogenesis at the level of IR-TK (Mol Endo 7: 1445-1455, 1993). In the present study, using recombinant alpha 2-HSG, which possesses 50-100 times the specific activity of plasma alpha 2-HSG, we have further investigated the molecular basis of this effect. We examined the insulin-stimulated Ras signalling pathway in Chinese Hamster Ovary cells overexpressing the human IR. alpha 2-HSG inhibits insulin-induced tyrosine phosphorylation of IRS-1 and the subsequent association of GRB2, as well as Sos, with IRS-1. This inhibition results in reduced guanine nucleotide exchange in p21ras. alpha 2-HSG also inhibits the stimulation of Raf phosphorylation, in response to insulin, leading to inhibition of MEK activity. In a parallel pathway, alpha 2-HSG also inhibits insulin-induced tyrosine phosphorylation of Shc. However, alpha 2-HSG does not affect any of the metabolic actions of insulin rested in these cells. These results suggest that, while insulin's mitogenic effects can be abolished by inhibition of insulin-induced IR-TK, propagation of signals for metabolic activities might utilize alternate of rescue mechanisms.
 ...
PMID:Recombinant human alpha 2-HS glycoprotein inhibits insulin-stimulated mitogenic pathway without affecting metabolic signalling in Chinese hamster ovary cells overexpressing the human insulin receptor. 911 49

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.
Mol Cell Biol 1997 Jul
PMID:Insulin-like growth factor I receptor signaling in transformation by src oncogenes. 919 8

To address whether Ras can be activated by insulin in the PC12 cell line, proteins interacting with insulin receptor and IRS-1 molecules and their tyrosine phosphorylation were analyzed by immunoblotting following immunoprecipitation with antibodies. Tyrosine phosphorylation of the insulin receptor and IRS-1 was increased by insulin. Grb2 and Ras-GAP appeared in the immunoprecipitates by anti-insulin receptor and anti-IRS-1 from insulin-treated cells. In addition, PI 3-kinase was activated by insulin treatment in this cell line and Grb2, Ras-GAP, and MAP kinase were coprecipitated with Ras from both insulin-treated and NGF-treated cells. Analysis of MAP kinases from insulin-treated cells revealed that insulin, like NGF, increased tyrosine phosphorylation. However, activation of the MAP kinase by NGF lasted longer than activation by insulin. These results indicate that Ras can be activated by insulin in the PC12 cell line and that Ras activation is neither an accurate nor a plausible method of discriminating signals between proliferation and differentiation.
Mol Cells 1997 Jun 30
PMID:Insulin activates Ras in the PC12 cell line. 926 35


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