Phorbol ester-mediated protein kinase C interaction with wild-type and COOH-terminal truncated insulin receptors.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin were compared in wild-type human insulin receptors (HIRc cells) and human insulin receptors lacking 43 COOH-terminal amino acid residues (HIR delta CT cells). TPA increased total phosphorylation of the wild-type insulin receptor and inhibited insulin-stimulated autophosphorylation by 32 +/- 10% in HIRc cells. TPA inhibited insulin-stimulated autophosphorylation by 46 +/- 14% in HIR delta CT cells and also caused a 65% decrease in basal phosphorylation. Insulin-stimulated tyrosine kinase activity for poly(Glu4/Tyr1) was inhibited by TPA in HIRc and HIR delta CT cells by 50 and 40%, respectively. TPA decreased insulin-stimulated glucose incorporation into glycogen by 50% in HIRc cells and to near basal levels in HIR delta CT cells; this inhibitory effect of TPA was reversed in both cell lines by staurosporine. In conclusion, 1) TPA-induced inhibition of insulin receptor tyrosine autophosphorylation was linked to concomitant inhibition of the biological effects of insulin in cells expressing either wild-type or COOH-terminal truncated insulin receptors; and 2) the inhibitory effects of TPA were not dependent upon phosphorylation of COOH-terminal residues and furthermore appeared to be independent of phosphorylation of any insulin receptor serine/threonine residues. These findings suggest a novel protein kinase C mechanism that results in altered insulin receptor function without increasing phosphorylation of the receptor.     

Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.     

Mutation of tyrosine 960 within the insulin receptor juxtamembrane domain impairs glucose transport but does not inhibit ligand-mediated phosphorylation of insulin receptor substrate-2 in 3T3-L1 adipocytes

CSF-1 is equipotent to insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960). CSF-1-treated adipocytes expressing the CSF1R/IRA960 were impaired in their ability to phosphorylate insulin receptor substrate 1 (IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting revealed that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for maximal stimulation of receptor-regulated glucose transport or glycogen synthesis.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.     

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.     

Interaction of guanylyl cyclase C with SH3 domain of Src tyrosine kinase. Yet another mechanism for desensitization

Protein-protein interactions mediated by the Src homology 3 (SH3) domain have been implicated in the regulation of receptor functions for subcellular localization of proteins and the reorganization of cytoskeleton. The experiments described in this article begin to identify the interaction of the SH3 domain of Src tyrosine kinase with the guanylyl cyclase C receptor after activation with Escherichia coli heat-stable enterotoxin (ST). Only one of two post-translationally modified forms of guanylyl cyclase C from T84 colonic carcinoma cells bind to GST-SH3 fusion protein of Src and Hck tyrosine kinases. Interestingly, the GST-Src-SH3 fusion protein showed 2-fold more affinity to native guanylyl cyclase C in solution than the GST-Hck-SH3 fusion protein. The affinity of the GST-Src-SH3 fusion protein to guanylyl cyclase C increased on desensitization of receptor in vivo. An in vitro cyclase assay in the presence of GST-Src-SH3 fusion protein indicated inhibition of the catalytic activity of guanylyl cyclase C. The catalytic domain recombinant protein (GST-GCD) of guanylyl cyclase C could pull-down a 60-kDa protein that reacted with Src tyrosine antibody and also showed autophosphorylation. These data suggest that SH3 domain-mediated protein-protein interaction with the catalytic domain of guanylyl cyclase C inhibited the cyclase activity and that such an interaction, possibly mediated by Src tyrosine kinase or additional proteins, might be pivotal for the desensitization phenomenon of the guanylyl cyclase C receptor.     

Structural and functional characterization of insulin receptor substrate proteins and the molecular mechanisms of their interaction with insulin superfamily tyrosine kinase receptors and effector proteins

The literature data on the role of IRS1/IRS2 proteins, endogenous substrates for insulin receptor tyrosine kinase, in transduction of signals generated by insulin superfamily peptides (insulin, insulin-like growth factor) were analyzed. The molecular mechanisms of the functional coupling of IRS proteins with peptide receptors possessing a tyrosine kinase activity and SH2 domain-containing proteins (phosphatidylinositol 3-kinase, Grb2 adaptor protein, protein phosphotyrosine phosphatase) were discussed. The structural and functional properties of IRS proteins (distribution of functional domains and sites for tyrosine phosphorylation; conservatism of amino acid sequences) were characterized. The data on the alternative pathways of transduction of signals which are generated by insulin and related peptides and do not involve IRS proteins were analyzed. These pathways are realized through Shc proteins or via direct interaction between receptors and SH2 proteins. Amino acid sequences of IRS proteins and insulin superfamily tyrosine kinase receptors were compared. The homologous regions in IRS proteins and receptors, which can be responsible for their coupling with phosphatidylinositol 3-kinase and protein phosphotyrosine phosphatases, were identified.     

Overexpression of membrane glycoprotein PC-1 can influence insulin action at a post-receptor site

An elevated content of membrane glycoprotein PC-1 has been observed in cells and tissues of insulin resistant patients. In addition, in vitro overexpression of PC-1 in cultured cells induces insulin resistance associated with diminished insulin receptor tyrosine kinase activity. We now find that PC-1 overexpression also influences insulin receptor signaling at a step downstream of insulin receptor tyrosine kinase, independent of insulin receptor tyrosine kinase. In the present studies, we employed Chinese hamster ovary cells that overexpress the human insulin receptor (CHO IR cells; ∼10⁶ receptors per cell), and transfected them with human PC-1 c-DNA (CHO IR PC-1). In CHO IR PC-1 cells, insulin receptor tyrosine kinase activity was unchanged, following insulin treatment of cells. However, several biological effects of insulin, including glucose and amino acid uptake, were decreased. In CHO IR PC-1 cells, insulin stimulation of mitogen-activated protein (MAP) kinase activity was normal, suggesting that PC-1 overexpression did not affect insulin receptor activation of Ras, which is upstream of MAP kinase. Also, insulin-stimulated phosphatidylinositol (PI)-3-kinase activity was normal, suggesting that PC-1 overexpression did not interfere with the activation of this enzyme by insulin receptor substrate-1. In these cells, however, insulin stimulation of p70 ribosomal S6 kinase activity was diminished. These studies suggest, therefore, that, in addition to blocking insulin receptor tyrosine kinase activation, PC-1 can also block insulin receptor signaling at a post-receptor site. J. Cell. Biochem. 68:366–377, 1998. © 1998 Wiley-Liss, Inc.     

Cellular chromium enhances activation of insulin receptor kinase

Chromium has been recognized for decades as a nutritional factor that improves glucose tolerance by enhancing in vivo insulin action, but the molecular mechanism is unknown. Here we report pretreatment of CHO-IR cells with chromium enhances tyrosine phosphorylation of the insulin receptor. Different chromium(III) compounds were effective at enhancing insulin receptor phosphorylation in intact cells, but did not directly activate recombinant insulin receptor kinase. The level of insulin receptor phosphorylation in cells can be increased by inhibition of the opposing protein tyrosine phosphatase (PTP1B), a target for drug development. However, chromium did not inhibit recombinant human PTP1B using either p-nitrophenyl phosphate or the tyrosine-phosphorylated insulin receptor as the substrate. Chromium also did not alter reversible redox regulation of PTP1B. Purified plasma membranes exhibited insulin-dependent kinase activity in assays using substrate peptides mimicking sites of Tyr phosphorylation in the endogenous substrate IRS-1. Plasma membranes prepared from chromium-treated cells had higher specific activity of insulin-dependent kinase relative to controls. We conclude that cellular chromium potentiates insulin signaling by increasing insulin receptor kinase activity, separate from inhibition of PTPase. Our results suggest that nutritional and pharmacological therapies may complement one another to combat insulin resistance, a hallmark of type 2 diabetes.     

Alterations in the tyrosine kinase activity of the insulin receptor produced by in vitro hyperinsulinemia

The tyrosine kinase activity of the insulin receptor was examined in fat cells made insulin resistant by hyperinsulinemia. Adipocytes previously treated for 16 h in vitro with 0.1 microM insulin were markedly insensitive to insulin as shown by the requirement of 2.3-fold higher concentration of insulin to stimulate half-maximal activation of glucose metabolism (glucose oxidation and glucose conversion to lipids). A similar rightward shift in the insulin dose-response curve was also evident for an insulin action not dependent on glucose metabolism, i.e. the inhibition of hormone-stimulated lipolysis. The almost 60% loss of insulin sensitivity occurred in conjunction with a 37% loss in insulin-binding activity, an alteration caused by a reduction in the number of insulin receptors. Studies of tyrosine kinase activity demonstrated a preferential alteration in this insulin receptor property, in addition to the receptor loss. The insulin-stimulated Vmax for the phosphorylation of the artificial substrate, Glu80-Tyr20, was significantly reduced by almost 40%, when the activity was expressed per insulin binding and measured in a receptor preparation purified by wheat germ affinity chromatography and immunoprecipitation. An elevation in basal tyrosine kinase activity was also present, which correlated with a lower apparent Km for ATP (0.025 mM) in comparison to the Km of 0.070 mM for the receptors from control cells. These findings indicate the presence of two types of alterations that involve the insulin receptor and hyperinsulinemia: one constituted by a modest reduction in receptor number and a second by modifications in the tyrosine kinase activity of the remaining receptors. Both alterations are required to explain the magnitude of the insulin resistance that develops after 16 h of insulin treatment.     

Inhibition of the insulin receptor tyrosine kinase by sphingosine.

Sphingosine inhibits autophosphorylation of the insulin receptor tyrosine kinase in vitro and in situ. This lysosphingolipid has been shown previously to inhibit the Ca2+/lipid-dependent protein kinase C. Here we show that insulin-dependent autophosphorylation of partially purified insulin receptor is half-maximally inhibited by 145 microM sphingosine (9 mol %) in Triton X-100 micelles. Half-maximal inhibition of protein kinase C autophosphorylation occurs with 60 microM sphingosine (3.4 mol %) in Triton X-100 mixed micelles containing phosphatidylserine and diacylglycerol. Sphingomyelin does not inhibit significantly the insulin receptor, suggesting that, as with protein kinase C, the free amino group may be essential for inhibition. Similar to the effects observed for protein kinase C, inhibition of the insulin receptor kinase by sphingosine is reduced in the presence of other lipids. However, the reduction displays a marked dependence on the lipid species: phosphatidylserine, but not a mixture of lipids compositionally similar to the cell membrane, markedly reduces the potency of sphingosine inhibition. The inhibition occurs at the level of the protein/membrane interaction: a soluble form of the insulin receptor comprising the cytoplasmic kinase domain is resistant to sphingosine inhibition. Lastly, sphingosine inhibits the insulin-stimulated rate of tyrosine phosphorylation of the insulin receptor in NIH 3T3 cells expressing the human insulin receptor. These results suggest that sphingosine alters membrane function independently of protein kinase C.     

Crystal structure of a complex between protein tyrosine phosphatase 1B and the insulin receptor tyrosine kinase

Protein tyrosine phosphatase 1B (PTP1B) is a highly specific negative regulator of insulin receptor signaling in vivo. The determinants of PTP1B specificity for the insulin receptor versus other receptor tyrosine kinases are largely unknown. Here, we report a crystal structure at 2.3 A resolution of the catalytic domain of PTP1B (trapping mutant) in complex with the phosphorylated tyrosine kinase domain of the insulin receptor (IRK). The crystallographic asymmetric unit contains two PTP1B-IRK complexes that interact through an IRK dimer interface. Rather than binding to a phosphotyrosine in the IRK activation loop, PTP1B binds instead to the opposite side of the kinase domain, with the phosphorylated activation loops sequestered within the IRK dimer. The crystal structure provides evidence for a noncatalytic mode of interaction between PTP1B and IRK, which could be important for the selective recruitment of PTP1B to the insulin receptor.     

Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation.

The tyrosine kinase activity of the epidermal growth factor (EGF) receptor is regulated by a truncated receptor of 100 kilodaltons (kDa) that contains the EGF-binding site but not the kinase domain. The inhibition of kinase is not due to competition for available EGF or for the kinase substrate-binding site. Chemical cross-linking studies suggest that the 100-kDa receptor may form a heterodimer with the intact EGF receptor. Structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the Neu receptor, were not inhibited by the 100-kDa receptor. The results indicate that (i) the inhibition was specific for the EGF receptor, (ii) the kinase domain had little or no role in determining target specificity, and (iii) the regulation of kinase may be due to a specific interaction of the 100-kDa receptor with the ligand-binding domain of the EGF receptor kinase.     

beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis

beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2-adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta-arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.     

In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor. Effects of fatty acid on receptor kinase and substrate phosphorylation.

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor beta-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr19, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (KIR0.5) was identical to that necessary for half-maximal ALBP phosphorylation (KALBP0.5), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the Vmax (to 0.33 fmol/min/fmol insulin-binding sites) while the Km for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor beta-subunit, the presence of oleate bound to ALBP increased the kcat/Km greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I0.5 approximately 4 microM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 approximately 190 microM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.     

Inhibition of the insulin receptor tyrosine kinase by phosphatidic acid

The lipid second messenger, phosphatidic acid, inhibits the intrinsic tyrosine kinase activity of the insulin receptor in detergent-lipid mixed micelles or in reconstituted membranes. Enzymatic studies revealed that this lipid second messenger inhibits the catalytic activity of partially purified insulin receptor without affecting the affinity of the receptor for insulin. Selectivity in the protein-lipid interaction is suggested by the inability of several other acidic lipids to affect the kinase activity of the receptor and by the relative insensitivity of the inhibition to increasing ionic strength and, in some cases, micelle surface charge. Lysophosphatidic acid and phosphatidic acids with short acyl chains do not affect significantly the receptor's kinase activity, suggesting that hydrophobic interactions are involved in the inhibition. Thus, both a high affinity interaction of the insulin receptor with the phosphate headgroup and a stabilizing hydrophobic interaction with the acyl chains contribute to the inhibitory protein-lipid interaction. The selective sensitivity of the insulin receptor to phosphatidic acid suggests that the receptor-mediated generation of this lipid in the plasma membrane could negatively modulate insulin receptor function. © 1996 Wiley-Liss, Inc.     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

Mutational analysis of the interaction between insulin receptor and IGF-I receptor with c-Crk and Crk-L in a yeast two-hybrid system

The SH2/SH3 adapter proteins of the Crk family are potent signal transducers after receptor tyrosine kinase stimulation with insulin or IGF-1. We have employed a yeast two-hybrid approach and mutational analysis to dissect the capabilities of the insulin receptor and the IGF-I receptor to directly associate with Crk isoforms. Insulin receptor stably recruits full length Crk by association with its SH2 domain in an auto-phosphorylation dependent manner. In contrast, interaction of the IGF-I receptor with the Crk-IISH2 domain was only detectable when Crk-II was truncated in its C-terminal part, indicating the transient nature of this interaction. From these data it can be concluded that members of the insulin receptor family activate Crk proteins in a differential manner.     

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.