Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aim of this study was to compare the effects of insulin and the insulinomimetic agent, englitazone, on functional end points and putative mediators of insulin action in 3T3-L1 adipocytes. Cells were incubated with englitazone for 48 h or with insulin for 10 or 30 min, or both, and 2-deoxy-D-[3H]glucose (2DG) uptake and lipogenesis (from [14C]glucose) were measured. Tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and pp60, and phosphatidylinositol (PI) 3-kinase activity (using PI as substrate) and mitogen-activated protein kinase (MAPK) activity were assayed in cell lysates. Englitazone increased 2DG uptake in a concentration-dependent (10-100 micromol/l) manner by up to sixfold, and preincubation with englitazone significantly enhanced insulin-stimulated 2DG uptake. However, englitazone had a biphasic effect on lipogenesis (163 +/- 13% basal at 10 micromol/l vs. 96 +/- 14% at 100 micromol/l), but when acetate was used as substrate, only concentration-dependent inhibition of lipogenesis occurred. In addition, englitazone decreased insulin-stimulated lipogenesis in a concentration-dependent manner. Englitazone did not increase IR, IRS-1/IRS-2, pp60, or MAPK phosphorylation, nor did it enhance insulin's stimulation of these parameters. Although englitazone alone did not activate PI 3-kinase, it did enhance the stimulation of the enzyme produced by a submaximally effective insulin concentration. Significant (63%) inhibition of insulin-stimulated lipogenesis occurred at a concentration of englitazone (30 micromol/l) that did not affect MAPK activation, which suggests that the drug's inhibitory effect on lipogenesis is not mediated by this pathway. Englitazone did not affect the expression of the peroxisome proliferator response element-containing fatty acyl CoA synthase gene, although it cannot be ruled out that expression of other lipogenic enzymes are altered by englitazone via peroxisome proliferator activated receptor-gamma activation or by an alternate pathway. Thus englitazone stimulates 2DG uptake without affecting PI 3-kinase, but it can enhance both insulin-stimulated 2DG uptake and PI 3-kinase activity. However, englitazone inhibits insulin-stimulated lipogenesis without inhibiting PI 3-kinase activity. Assuming activation of PI 3-kinase mediates insulin-stimulated 2-DG and lipogenesis, then the signaling pathways for each process diverge beyond PI 3-kinase.
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PMID:Possibility of distinct insulin-signaling pathways beyond phosphatidylinositol 3-kinase-mediating glucose transport and lipogenesis. 951 10

The monomeric insulin analogue insulin lispro (Lys B28, Pro B29) is a rapid-acting insulin with a shorter duration of activity than human regular insulin. This compound has the advantage of reducing early postprandial hyperglycemia and the accompanying late hypoglycemia, thereby improving overall blood glucose control. To date, all published studies of the functional properties of insulin lispro have been conducted in whole animals. This study aimed to characterize the cellular actions of insulin lispro and the signals it elicits in an insulin-sensitive muscle cell line, L6 cells. Comparing the cellular actions of insulin lispro with those of human regular insulin, a number of observations were made. (1) Insulin lispro stimulated glucose and amino acid transport into L6 myotubes with a dose dependency and time course virtually identical to those of human regular insulin. (2) Insulin lispro was as effective as human regular insulin in stimulating time-dependent phosphorylation of insulin receptor substrate 1 (IRS-1), p70 ribosomal S6 kinase, and two isoforms of mitogen-activated protein kinase (ERK1 and ERK2). (3) Insulin lispro's ability to induce the association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase was similar to that of human regular insulin. (4) As with human regular insulin, 100 nmol of the fungal metabolite wortmannin completely inhibited insulin lispro stimulation of glucose uptake. We concluded that the cellular actions of insulin lispro are similar to those of human regular insulin with respect to glucose and amino acid uptake and that the biochemical signals elicited are also comparable.
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PMID:Stimulation of glucose and amino acid transport and activation of the insulin signaling pathways by insulin lispro in L6 skeletal muscle cells. 952 10

Because insulin-like growth factor-I (IGF-I), insulin, and interleukin-4 (IL-4) have known biological effects in breast cancer cells and signal through insulin-receptor substrate (IRS) adaptor proteins, we examined the expression and function of IRS-1 and IRS-2 in breast tumors and cell lines. IRS-1 and IRS-2 were expressed by cell lines and primary breast tumor specimens. IGF-I, insulin, and IL-4 treatment of MCF-7 and ZR-75, and IGF-I treatment of T47-D breast cancer cells, resulted in much greater tyrosine phosphorylation of IRS-1 compared with IRS-2. Furthermore, IGF-I stimulated greater tyrosine phosphorylation of IRS-1 than either insulin or IL-4. IGF-I treatment also enhanced association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with IRS-1 and stimulated increased enzymatic activity compared with IL-4 and insulin in all three cell lines. Similarly, mitogen-activated protein kinase activity was greater in IGF-I-stimulated cells. To determine the functional significance of the activation of these pathways, we inhibited activation of phosphatidylinositol 3-kinase with wortmannin and mitogen-activated protein kinase with PD098059. Both compounds inhibited IGF-stimulated growth, suggesting that both pathways contributed to the mitogenic response to IGF-I. We conclude that IRS-1, and not IRS-2, is the predominant signaling molecule activated by IGF-I, insulin, and IL-4. Furthermore, enhanced tyrosine phosphorylation of IRS-1 by IGF-I, compared with either insulin or IL-4, is associated with greater activation of mitogenic downstream signaling pathways resulting in enhanced cell growth.
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PMID:Insulin receptor substrate-1 is the predominant signaling molecule activated by insulin-like growth factor-I, insulin, and interleukin-4 in estrogen receptor-positive human breast cancer cells. 954 45

We have used the expression of muscarinic m1 receptors in the preadipocytic 3T3-L1 cell line for dissecting the nature of the G protein-linked pathways governing adipocytic differentiation, a complex process controlled by many stimuli and their downstream targets. 3T3-L1 cells can be differentiated by insulin or by ras oncogenes, and MAP kinase has been implicated in this process. However, m1 stimulation failed to induce differentiation of 3T3-L1 cells. Furthermore, it prevented insulin or v-ras-induced adipocytic differentiation, utilizing a protein kinase C-independent pathway. m1 stimulation did not alter the phosphorylation state of the insulin receptor substrates IRS-1 and SHC, nor the recruitment of Grb-2. Interestingly, whereas m1 receptors potently activated MAP kinase, another differentiation-inhibitor, TNF alpha, did not affect it. These results suggest that the control of adipocytic differentiation can occur utilizing a biochemical route independent of protein kinase C, and acting downstream, or independently from the Ras-MAP kinase pathway.
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PMID:Transforming G protein-coupled receptors block insulin and ras-induced adipocytic differentiation in 3T3-L1 cells: evidence for a PKC and MAP kinase independent pathway. 957 Nov 94

Salts of the trace element vanadium, such as sodium orthovanadate and vanadyl sulfate (VS), exhibit a myriad of insulin-like effects, including stimulation of glycogen synthesis and improvement of glucose homeostasis in type I and type II animal models of diabetes mellitus. However, the cellular mechanism by which these effects are mediated remains poorly characterized. We have shown earlier that different vanadium salts stimulate the MAP kinase pathway and ribosomal-S-6-kinase (p70s6k) in chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) [Pandey, S. K., Chiasson, J.-L., and Srivastava, A. K. (1995) Mol. Cell. Biochem. 153, 69-78]. In the present studies, we have investigated if similar to insulin, VS also activates phosphatidylinositol 3-kinase (PI3-k) activity, and whether VS-induced activation of the PI3-k, MAP kinase, and p70s6k pathways contributes to glycogen synthesis. Treatment of CHO-HIR cells with VS resulted in increased glycogen synthesis and PI3-k activity which were blocked by pretreatment of the cells with wortmannin and LY294002, two specific inhibitors of PI3-k. On the other hand, PD98059 and rapamycin, specific inhibitors of the MAP kinase pathway and p70s6k, respectively, were unable to inhibit VS-stimulated glycogen synthesis. Moreover, VS-stimulated glycogen synthesis and PI3-k were observed without any change in the tyrosine phosphorylation of insulin receptor (IR) beta-subunit but were associated with increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In addition, PI3-k activation was detected in IRS-1 immunoprecipitates from VS-stimulated cells, indicating that tyrosine-phosphorylated IRS-1 was able to interact and thereby activate PI3-k in response to VS. Taken together, these results provide evidence that tyrosine phosphorylation of IRS-1 and activation of PI3-k play a key role in mediating the insulinomimetic effect of VS on glycogen synthesis independent of IR-tyrosine phosphorylation.
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PMID:Vanadyl sulfate-stimulated glycogen synthesis is associated with activation of phosphatidylinositol 3-kinase and is independent of insulin receptor tyrosine phosphorylation. 957 88

In the present study we have investigated the contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling pathways in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulated IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation was barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha revealed a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain showed functional differences. While a strong association between IRS-1/Grb-2 was found, IRS-2/Grb-2 association was virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increased the amount of Grb-2-associated SHC proteins by the same extent. Our results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation that leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2 leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation.
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PMID:Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes. 960 31

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.
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PMID:Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase. 962 69

PRL is an anterior pituitary hormone that, along with GH and PLs, forms a family of hormones that probably resulted from the duplication of an ancestral gene. The PRLR is also a member of a larger family, known as the cytokine class-1 receptor superfamily, which currently has more than 20 different members. PRLRs or binding sites are widely distributed throughout the body. In fact, it is difficult to find a tissue that does not express any PRLR mRNA or protein. In agreement with this wide distribution of receptors is the fact that now more than 300 separate actions of PRL have been reported in various vertebrates, including effects on water and salt balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and immune regulation and protection. Clearly, a large proportion of these actions are directly or indirectly associated with the process of reproduction, including many behavioral effects. PRL is also becoming well known as an important regulator of immune function. A number of disease states, including the growth of different forms of cancer as well as various autoimmune diseases, appear to be related to an overproduction of PRL, which may act in an endocrine, autocrine, or paracrine manner, or via an increased sensitivity to the hormone. The first step in the mechanism of action of PRL is the binding to a cell surface receptor. The ligand binds in a two-step process in which site 1 on PRL binds to one receptor molecule, after which a second receptor molecule binds to site 2 on the hormone, forming a homodimer consisting of one molecule of PRL and two molecules of receptor. The PRLR contains no intrinsic tyrosine kinase cytoplasmic domain but associates with a cytoplasmic tyrosine kinase, JAK2. Dimerization of the receptor induces tyrosine phosphorylation and activation of the JAK kinase followed by phosphorylation of the receptor. Other receptor-associated kinases of the Src family have also been shown to be activated by PRL. One major pathway of signaling involves phosphorylation of cytoplasmic State proteins, which themselves dimerize and translocate to nucleus and bind to specific promoter elements on PRL-responsive genes. In addition, the Ras/Raf/MAP kinase pathway is also activated by PRL and may be involved in the proliferative effects of the hormone. Finally, a number of other potential mediators have been identified, including IRS-1, PI-3 kinase, SHP-2, PLC gamma, PKC, and intracellular Ca2+. The technique of gene targeting in mice has been used to develop the first experimental model in which the effect of the complete absence of any lactogen or PRL-mediated effects can be studied. Heterozygous (+/-) females show almost complete failure to lactate after the first, but not subsequent, pregnancies. Homozygous (-/-) females are infertile due to multiple reproductive abnormalities, including ovulation of premeiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Twenty per cent of the homozygous males showed delayed fertility. Other phenotypes, including effects on the immune system and bone, are currently being examined. It is clear that there are multiple actions associated with PRL. It will be important to correlate known effects with local production of PRL to differentiate classic endocrine from autocrine/paracrine effects. The fact that extrapituitary PRL can, under some circumstances, compensate for pituitary PRL raises the interesting possibility that there may be effects of PRL other than those originally observed in hypophysectomized rats. The PRLR knockout mouse model should be an interesting system by which to look for effects activated only by PRL or other lactogenic hormones. On the other hand, many of the effects reported in this review may be shared with other hormones, cytokines, or growth factors and thus will be more difficult to study. (ABSTRACT TRUNCATED)
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PMID:Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. 962 54

We have studied the involvement of murine c-Crk, an SH2/SH3 containing adaptor protein, in signaling pathways stimulated by different receptor tyrosine kinases. We show here that c-Crk is associated with components of insulin- and PDGF-dependent signaling pathways. Insulin treatment of murine myoblast cells induces the formation of stable complex of endogenous c-Crk with insulin receptor substrate-1 (IRS-1) mediated via the SH2 domain of Crk. The ligand dependent physical association of c-Crk with IRS-1 is direct. However IRS-1 is also co-precipitated with c-Crk from quiescent L6 cells. The association of IRS-1 with c-Crk in quiescent cells is probably not direct since Far Western blot analysis did not reveal the binding of neither SH2 domain nor amino-terminal SH3 domain of c-Crk to IRS-1 from unstimulated cells. We also show that PDGF treatment of murine myoblast cells induces association of c-Crk with the PDGF receptor and tyrosine phosphorylation of c-Crk. Overexpression of c-Crk enhanced insulin- but not PDGF-induced activation of MAP kinases when compared to parental cell lines. Thus, the formation of the direct IRS-1/Crk complex appears to be crucial for Crk-mediated insulin-induced activation of MAP kinase, whereas Crk is probably involved in other PDGF-induced responses. These data provide support to the hypothesis that insulin and PDGF employ different mechanisms for activation of MAP kinase cascade.
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PMID:Crk protein binds to PDGF receptor and insulin receptor substrate-1 with different modulating effects on PDGF- and insulin-dependent signaling pathways. 962 9

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.
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PMID:Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways. 963 70


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