Negative regulation of insulin-stimulated mitogen-activated protein kinase signaling by Grb10

Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.     

Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.     

Hormonal regulation of the Grb14 signal modulator and its role in cell cycle progression of MCF-7 human breast cancer cells

Growth factor receptor bound (Grb)14 is a member of the Grb7 family of src homology (SH)2 domain-containing proteins. These proteins perform both adaptor and modulatory roles in receptor tyrosine kinase (RTK) signaling, although their regulation is poorly understood. In this study, a positive correlation between Grb14 protein expression and ER alpha status in breast cancer cell lines led us to investigate regulation of Grb14 by estradiol and insulin, which synergize in the regulation of breast cancer cell proliferation. In MCF-7 cells maintained in charcoal-stripped serum, Grb14 expression was downregulated by estradiol and increased by the pure anti-estrogen ICI 182780. Under serum-free conditions, insulin enhanced Grb14 expression but this effect was repressed by estradiol when both hormones were used in combination. Using a system in which c-Myc induction drives cell cycle progression independently of estradiol, we demonstrated that Grb14 regulation was specific to estradiol treatment. Finally, we demonstrated a novel functional role for Grb14 whereby its overexpression inhibited not only insulin- but also estrogen-induced cell cycle progression. This was associated with decreased extracellular signal-regulated kinase (Erk)1/2 activation in insulin-stimulated Grb14-overexpressing cells. These data represent the first demonstration of regulation of Grb14 expression levels in response to hormonal stimuli, and are consistent with its role as a repressor of insulin signaling where it is induced as a negative feedback mechanism. A role for Grb14 is also shown in estrogen/insulin crosstalk since estradiol blocks the insulin-induced induction of this protein.     

Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.     

Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.     

Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.     

Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice

Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18-22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased beta-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

Insulin-stimulated cell growth in insulin receptor substrate-1–deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase–independent pathway

In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21^ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells). The PI3-K inhibitor LY294002 (50 μM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 μM) consistently reduced insulin-dependent growth in all three cell lines. Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K–dependent or–independent pathways. J. Cell. Biochem. 70:268–280, 1998. © 1998 Wiley-Liss, Inc.     

Meg1/Grb10 overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the IGF1R and IR cascades

The Meg1/Grb10 protein has been implicated as an adapter protein in the signaling pathways from insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in vitro. To elucidate its in vivo function, four independent Meg1/Grb10 transgenic mouse lines were established, and the effects of excess Meg1/Grb10 on both postnatal growth and glucose metabolism were examined. All of the Meg1/Grb10 transgenic mice showed growth retardation after weaning (3-4 weeks), which indicates that ectopic overexpression of Meg1/Grb10 inhibits postnatal growth that is mediated by IGF1 via IGF1R. In addition, the mice became hyperinsulinemic owing to high levels of insulin resistance, which demonstrates that Meg1/Grb10 also modulates the insulin receptor cascade negatively in vivo. Type II diabetes arose frequently in the two transgenic lines, which also showed impaired glucose tolerance. In these mice, severe atrophy of the pancreatic acinus cells was associated with high-level production of Meg1/Grb10 in the pancreas. These results suggest that Meg1/Grb10 inhibits the function of both insulin and IGF1 receptors in these cells, since a similar phenotype has been reported for Ir and Igf1r double knockout mice. Taken together, these results indicate that Meg1/Grb10 interacts with both insulin and IGF1 receptors in vivo, and negatively regulates the IGF growth pathways via these receptors.     

Inhibition of insulin receptor catalytic activity by the molecular adapter Grb14.

Grb14 belongs to the Grb7 family of adapters and was recently identified as a partner of the insulin receptor (IR). Here we show that Grb14 inhibits in vitro IR substrate phosphorylation. Grb14 does not alter the K(m) for ATP and behaves as an uncompetitive inhibitor for the IR substrate. Similar experiments performed with other members of the Grb7 family, Grb7 and Grb10, and with IGF-1 receptor argue in favor of a specific inhibition of the IR catalytic activity by Grb14. The IR-interacting domain of Grb14, the PIR, is sufficient for the inhibitory effect of Grb14, whereas the SH2 domain has no effect on IR catalytic activity. In Chinese hamster ovary (CHO) cells overexpressing both IR and Grb14, Grb14 binds to the IR as early as 1 min after insulin stimulation, and the two proteins remain associated. When interacting with Grb14, the IR is protected against tyrosine phosphatases action and therefore maintained under a phosphorylated state. However, the binding of Grb14 to the IR induces an early delay in the activation of Akt and ERK1/2 in CHO-IR cells, and ERK1/2 are less efficiently phosphorylated. These findings show that Grb14 is a direct inhibitor of the IR catalytic activity and could be considered as a modulator of insulin signaling.     

Grb10, a Positive, Stimulatory Signaling Adapter in Platelet-Derived Growth Factor BB-, Insulin-Like Growth Factor I-, and Insulin-Mediated Mitogenesis

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR). Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor beta (PDGFRbeta), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGRbeta site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Grb10 expression from cDNA with an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth factor (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGF-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable Grb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transient overexpression of complete Grb10 increased whereas cell-permeable Grb10 SH2 domain fusion peptides substantially decreased the cell proliferation rate (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimulatory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. This function appears to involve the Grb10 SH2 domain, a novel sequence termed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and insulin-mediated mitogenesis. In contrast, PDGF-BB-mediated mitogenesis appears to depend on the SH2 but not on the Pro-rich region and may involve other, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.     

Effect of phosphotyrosyl-IRS-1 level and insulin receptor tyrosine kinase activity on insulin-stimulated phosphatidylinositol 3, MAP,and S6 kinase activities

The role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS-1, the insulin receptor, or both IRS-1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS-1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin-stimulated IRS-1 associated Ptdlns 3′-kinase activit in these cells was 1-, 1.5-, 3-, and 3-fold, while insulin sensitivity, as determined by ED₅₀, was 1-, 2.5-, 10-, and 10-fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl-IRS-1; however, the increased level of phosphotyrosyl-IRS-1 seen in CHO/IR/IRS-1 cells did not further increase these responses. Likewise, maximal insulin-stimulated MAP kinase activity in these cell lines increased in parallel with IRS-1 tyrosine phosphorylation except in the CHO/IR/IRS-1 cell lines with activity levels of one-, five-, nine-, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin-stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl-IRS-1, but an increase was observed with insulin-stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS-1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS-1 and other possible cellular substrates. © 1995 Wiley-Liss, Inc.     

Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.     

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.     

Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation

Background

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14?

Results

Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2^Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line.

Conclusions

Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling.

     

Reduction of protein-tyrosine phosphatase-1B increases insulin signaling in FAO hepatoma cells

Protein-tyrosine phosphatase-1B (PTP1B) has been implicated as a negative regulator of insulin signaling. PTP1B dephosphorylates the insulin receptor and insulin receptor substrates (IRS-1/2), inhibiting the insulin-signaling pathway. PTP1B has been reported to be elevated in diabetes and insulin-resistant states. Conversely, PTP1B null mice have increased insulin sensitivity. To further investigate the effect of PTP1B reduction on insulin signaling, FAO rat hepatoma cells were transfected, by electroporation, with a specific PTP1B antisense oligonucleotide (ASO), or a control oligonucleotide. The PTP1B ASO caused a 50-70% reduction in PTP1B protein expression as measured by Western blot analysis. Upon insulin stimulation, an increase in the phosphorylation of the insulin receptor and insulin receptor substrates was observed, without any change in protein expression levels. Reduction of PTP1B expression in FAO cells also caused an increase in insulin-stimulated phosphorylation of PKB and GSK3, without any change in protein expression. These results demonstrate that reduction of PTP1B can modulate key insulin signaling events downstream of the insulin receptor.