Epidermal growth factor and transforming growth factor alpha mimic the effects of insulin in human fat cells and augment downstream signaling in insulin resistance

The ability of the growth factors epidermal growth factor (EGF), transforming growth factor alpha, and platelet-derived growth factor to exert insulin-like effects on glucose transport and lipolysis were examined in human and rat fat cells. No effects were found in rat fat cells, whereas EGF (EC(50) for glucose transport approximately 0.02 nm) and transforming growth factor alpha (EC(50) approximately 0.2 nm), but not platelet-derived growth factor, mimicked the effects of insulin (EC(50) approximately 0.2 nm) on both pathways. EGF receptors, but not EGF, were abundantly expressed in human fat cells as well as in human skeletal muscle. EGF increased the tyrosine phosphorylation of several proteins (the EGF receptor, insulin receptor substrate (IRS)-1, IRS-2, and Grb2-associated binder 1), whereas Shc and Gab2 were only weakly and inconsistently phosphorylated. p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), was also found to associate with all of these docking molecules, showing that EGF activated PI 3-kinase pools that were additional to those of insulin. EGF and/or insulin increased protein kinase B/Akt serine phosphorylation to a similar extent, whereas mitogen-activated protein kinase phosphorylation was more pronounced for EGF than for insulin. The impaired insulin-stimulated downstream signaling, measured as protein kinase B/Akt serine phosphorylation, in insulin-resistant cells (Type 2 diabetes) was improved by the addition of EGF. Thus, EGF receptors, but not EGF, are abundantly expressed in human fat cells and skeletal muscle. EGF mimics the effects of insulin on both the metabolic and mitogenic pathways but utilize in part different signaling pathways. Both insulin and EGF increase the tyrosine phosphorylation and activation of IRS-1 and IRS-2, whereas EGF is also capable of activating additional PI 3-kinase pools and, thus, can augment the downstream signaling of insulin in insulin-resistant states like Type 2 diabetes.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.     

Insulin receptor substrate 1 rescues insulin action in CHO cells expressing mutant insulin receptors that lack a juxtamembrane NPXY motif.

Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK.     

Immobilization depresses insulin signaling in skeletal muscle

Prolonged immobilization depresses insulin-induced glucose transport in skeletal muscle and leads to a catabolic state in the affected areas, with resultant muscle wasting. To elucidate the altered intracellular mechanisms involved in the insulin resistance, we examined insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit (IR-beta) and insulin receptor substrate (IRS)-1 and activation of its further downstream molecule, phosphatidylinositol 3-kinase (PI 3-K), after unilateral hindlimb immobilization in the rat. The contralateral hindlimb served as control. After 7 days of immobilization of the rat, insulin was injected into the portal vein, and tibialis anterior muscles on both sides were extracted. Immobilization reduced insulin-stimulated tyrosine phosphorylation of IR-beta and IRS-1. Insulin-stimulated binding of IRS-1 to p85, the regulatory subunit of PI 3-K, and IRS-1-associated PI 3-K activity were also decreased in the immobilized hindlimb. Although IR-beta and p85 protein levels were unchanged, IRS-1 protein expression was downregulated by immobilization. Thus prolonged immobilization may cause depression of insulin-stimulated glucose transport in skeletal muscle by altering insulin action at multiple points, including the tyrosine phosphorylation, protein expression, and activation of essential components of insulin signaling pathways.     

Intracellular signalling pathways of okadaic acid leading to mitogenesis in Rat1 fibroblast overexpressing insulin receptors: okadaic acid regulates Shc phosphorylation by mechanisms independent of insulin

Okadaic acid is a powerful inhibitor of serine/threonine protein phosphatases 1 and 2A. Although it is known as a potent tumour promoter, the intracellular mechanism by which okadaic acid mediates its mitogenic effect remains to be clarified. We investigated the effect of okadaic acid on the activation of mitogenesis in Rat1 fibroblasts overexpressing insulin receptors. As previously reported, insulin induced Shc phosphorylation, Shc-Grb2 association, MAP kinase activation, and BrdU incorporation. Okadaic acid also stimulated tyrosine phosphorylation of Shc and its subsequent association with Grb2 in a time- and dose-dependent manner without affecting tyrosine phosphorylation of insulin receptor beta-subunit and IRS. However, to a lesser extent, okadaic acid stimulated MAP kinase activity and BrdU incorporation. Interestingly, preincubation of okadaic acid potentiated insulin stimulation of tyrosine phosphorylation of Shc (213% of control), Shc-Grb2 association (150%), MAP kinase activity (152%), and BrdU incorporation (148%). These results further confirmed the important role of Shc, but not IRS, in cell cycle progression in Rat1 fibroblasts. Furthermore, serine/ threonine phosphorylation appears to be involved in the regulation of Shc tyrosine phosphorylation leading to mitogenesis by mechanisms independent of insulin signalling.     

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.     

Receptor-isoform-selective insulin analogues give tissue-preferential effects

The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI) for inducing glycogen accumulation (75%) and lipogenesis (130%) than for affecting muscle (45%). For the same blood-glucose-lowering effect upon acute intravenous dosing of mice, INS-B gave a significantly higher degree of IR phosphorylation in liver than HI. These in vitro and in vivo results indicate that insulin analogues with IR-isoform-preferential binding affinity are able to elicit tissue-selective biological responses, depending on IR-A/IR-B expression.     

Insulin stimulation of the insulin receptor kinase can occur in the complete absence of beta subunit autophosphorylation

The glutamic acid:tyrosine (Glu:Tyr) synthetic polymer was observed to inhibit the insulin receptor beta subunit autophosphorylation with an IC50 of 0.20 mg/ml in the absence and 0.15 mg/ml in the presence of insulin. Even though complete blockade of beta subunit autophosphorylation was observed at 4.0 mg/ml Glu:Tyr, insulin was still capable of stimulating the exogenous protein kinase activity of the insulin receptor toward Glu:Tyr. Histone H2B (1.3 mg/ml) was also observed to inhibit the beta subunit autophosphorylation by approximately 80% with an IC50 of 0.31 and 0.35 mg/ml in the absence and presence of insulin, respectively. Similar to the results with Glu:Tyr, insulin was found to stimulate histone H2B phosphorylation under these conditions. Comparisons between the time courses of beta subunit autophosphorylation with those of Glu:Tyr phosphorylation both in the presence and absence of insulin confirmed that insulin can stimulate the exogenous protein kinase activity of the insulin receptor in the complete absence of beta subunit autophosphorylation. Prephosphorylation of the insulin receptor (from 0 to 1.3 mol of phosphate/mol of insulin receptor) in the absence of insulin was found to have no significant effect on the exogenous protein kinase activity when assayed both in the presence and absence of insulin. Insulin was observed to stimulate the phosphorylation of Glu:Tyr approximately 3-fold independent of the extent of beta subunit autophosphorylation. In contrast, prephosphorylation of the insulin receptors in the presence of insulin was observed to enhance the exogenous protein kinase activity dependent on the extent of autophosphorylation, such that by 1.4 mol of phosphate incorporated per mol of insulin receptor, insulin was found to maximally stimulate the initial rate of Glu:Tyr phosphorylation (approximately 9-fold). These results demonstrate that the insulin-dependent autophosphorylation of the insulin receptor results in an amplification of the insulin stimulation of the exogenous protein kinase activity, whereas the insulin-independent autophosphorylation does not.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Protein phosphatase 2A forms a molecular complex with Shc and regulates Shc tyrosine phosphorylation and downstream mitogenic signaling

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.     

Amiloride directly inhibits growth factor receptor tyrosine kinase activity

Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors.     

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras.

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.     

Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: Potential role of the NFkappa B pathway

The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO‐IR) and 3T3‐L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor β‐subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate‐1 (IRS‐1) on serine 307 (S307) in both CHO‐IR cells and 3T3‐L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin‐stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling‐1 and ‐3 (SOCS‐1 and ‐3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO‐IR cells were pre‐treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR β‐subunit and phosphorylation of IRS‐1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs. J. Cell. Biochem. 114: 1729–1737, 2013. © 2013 Wiley Periodicals, Inc.     

Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control

Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Str?lfors, P. (1999) FASEB. J. 13, 1961-1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with beta-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by (125)I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.     

In vitro endosome-lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

We have studied the endosome-lysosome transfer of internalized epidermal growth factor receptor (EGFR) complexes in a cell-free system from rat liver. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [(125)I]EGF was largely associated with a light endosomal fraction devoid of lysosomal markers. After an additional 30 min incubation in vitro in the presence of an ATP-regenerating system, the amount of [(125)I]EGF in this compartment decreased by 39%, with an increase in [(125)I]EGF in lysosomes. No transfer of [(125)I]EGF to the cytosol was detected. To assess the fate of the internalized EGFR protein over the time course of the endo-lysosomal transfer of the ligand, the effect of a saturating dose of native EGF on subsequent lysosomal targeting of the EGFR was evaluated by immunoblotting. A massive translocation of the EGFR to the endosomal compartment was observed in response to ligand injection coincident with its tyrosine phosphorylation and receptor recruitment of the tyrosine-phosphorylated adaptor protein Shc. During cell-free endosome-lysosome fusion, a time-dependent increase in the content of the EGFR and the two 55- and 46-kDa Shc isoforms was observed in lysosomal fractions with a time course superimposable with the lysosomal transfer of the ligand; no transfer of the 66-kDa Shc isoform was detected. The relationship between EGFR tyrosine kinase activity and EGFR sorting in endosomes investigated by immunoblot studies with anti-phosphotyrosine antibodies revealed that endosomal dephosphorylation of EGFR and Shc preceded lysosomal transfer. These results support the view that a lysosomal targeting machinery distinct from the endosomal receptor kinase activity, such as the recruitment of the signaling molecule Shc, may regulate this sorting event in the endosome.     

Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking.

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.