Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway

Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF did not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) were required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt was found to inhibit insulin signaling. Moreover, this inhibition required the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt were inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

Rapamycin promotes vascular smooth muscle cell differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling

The phenotypic plasticity of mature vascular smooth muscle cells (VSMCs) facilitates angiogenesis and wound healing, but VSCM dedifferentiation also contributes to vascular pathologies such as intimal hyperplasia. Insulin/insulin-like growth factor I (IGF-I) is unique among growth factors in promoting VSMC differentiation via preferential activation of phosphatidylinositol 3-kinase (PI3K) and Akt. We have previously reported that rapamycin promotes VSMC differentiation by inhibiting the mammalian target of rapamycin (mTOR) target S6K1. Here, we show that rapamycin activates Akt and induces contractile protein expression in human VSMC in an insulin-like growth factor I-dependent manner, by relieving S6K1-dependent negative regulation of insulin receptor substrate-1 (IRS-1). In skeletal muscle and adipocytes, rapamycin relieves mTOR/S6K1-dependent inhibitory phosphorylation of IRS-1, thus preventing IRS-1 degradation and enhancing PI3K activation. We report that this mechanism is functional in VSMCs and crucial for rapamycin-induced differentiation. Rapamycin inhibits S6K1-dependent IRS-1 serine phosphorylation, increases IRS-1 protein levels, and promotes association of tyrosine-phosphorylated IRS-1 with PI3K. A rapamycin-resistant S6K1 mutant prevents rapamycin-induced Akt activation and VSMC differentiation. Notably, we find that rapamycin selectively activates only the Akt2 isoform and that Akt2, but not Akt1, is sufficient to induce contractile protein expression. Akt2 is required for rapamycin-induced VSMC differentiation, whereas Akt1 appears to oppose contractile protein expression. The anti-restenotic effect of rapamycin in patients may be attributable to this unique pattern of PI3K effector regulation wherein anti-differentiation signals from S6K1 are inhibited, but pro-differentiation Akt2 activity is promoted through an IRS-1 feedback signaling mechanism.     

Regulation of insulin receptor substrate-1 expression levels by caveolin-1

The insulin receptor substrate-1 (IRS-1), a docking protein of the type 1 insulin-like growth factor receptor (IGF-IR) plays a significant role in cell proliferation and differentiation. The expression of IRS-1 is down-regulated in mouse embryo fibroblasts (MEFs) with a deletion of caveolin-1 (cav1) genes (KO cells). Levels of IRS-1 mRNA are not affected. Re-introduction of cav1 into KO cells rescues IRS-1 expression. Stabilization of protein levels is reciprocal and a strict correlation between IRS-1 and cav1 levels was confirmed in five cell lines, and in mouse tissues. IRS-1 binds through its phosphotyrosine binding (PTB) domain to tyrosine 14 (Y14) of cav1, the residue phosphorylated by IGF-1 stimulation and by v-src. The down-regulation of IRS-1 in cav-/- cells occurs via the proteasome pathway. These results indicate a novel mechanism for the regulation of IRS-1 expression levels, an important finding in view of IRS-1 role in cell proliferation and transformation.     

Impairment of bone healing by insulin receptor substrate-1 deficiency

Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1(-/-) mice). Among 15 IRS-1(-/-) mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1(-/-) fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1(-/-) chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1(-/-) culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.     

Nuclear translocation of insulin receptor substrate-1 by the insulin receptor in mouse embryo fibroblasts

Translocation of the insulin receptor substrate-1 (IRS-1) to the nuclei has been reported to occur in cells stimulated by insulin-like growth factor-1 (IGF-I) or expressing certain viral and cellular oncogenes. We show here that insulin can also induce nuclear translocation of IRS-1 in mouse embryo fibroblasts (MEF), that do not express the type 1 insulin-like growth factor receptor (IGF-IR). Only the A isoform of the insulin receptor (IR) can induce IRS-1 nuclear translocation, which is significant when the receptor is over-expressed. At physiological receptor levels, translocation occurs only in a fraction of cells, and only at high concentrations of ligand.     

Acetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation

Background  

Insulin receptor substrate (IRS) proteins are key moderators of insulin action. Their specific regulation determines downstream protein-protein interactions and confers specificity on growth factor signalling. Regulatory mechanisms that have been identified include phosphorylation of IRS proteins on tyrosine and serine residues and ubiquitination of lysine residues. This study investigated other potential molecular mechanisms of IRS-1 regulation.     

Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, Akt, NF-kappaB, and p300 pathway in microglia

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-kappaB inhibitor (pyrrolidine dithiocarbamate), IkappaB protease inhibitor (L-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IkappaBalpha phosphorylation inhibitor (Bay 117082), or NF-kappaB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IkappaB kinase alpha/IkappaB kinase beta, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Leptin-mediated an increase of IkappaB kinase alpha/IkappaB kinase beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-kappaB and p300 signaling pathway.     

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.     

Serine phosphorylation of insulin receptor substrate-1: a novel target for the reversal of insulin resistance.

Insulin resistance, the failure to respond to normal circulating concentrations of insulin, is a common state associated with obesity, aging, and a sedentary lifestyle. Compelling evidence implicates TNFalpha as the cause and link between obesity and insulin resistance. Serine phosphorylation of insulin receptor substrate-1 seems prominent among the mechanisms of TNFalpha-induced insulin resistance. Recent advances indicate that serine kinases may phosphorylate and thus inhibit the tyrosine phosphorylation of insulin receptor substrate-1, revealing an integration point of TNFalpha and insulin signaling pathways. Selective targeting of the molecular scenery whereby this key phosphorylation occurs/operates represents a rich area for the development of rationally designed new antidiabetic drugs. In relation to efficacy and side effects, this prospect should permit a more precise and perhaps individualized approach to therapeutic intervention, allowing clinicians to focus the attack where the problem lies.     

Functional studies of Akt isoform specificity in skeletal muscle in vivo; maintained insulin sensitivity despite reduced insulin receptor substrate-1 expression

The phosphoinositide 3-kinase/Akt pathway is thought to be essential for normal insulin action and glucose metabolism in skeletal muscle and has been shown to be dysregulated in insulin resistance. However, the specific roles of and signaling pathways triggered by Akt isoforms have not been fully assessed in muscle in vivo. We overexpressed constitutively active (ca-) Akt-1 or Akt-2 constructs in muscle using in vivo electrotransfer and, after 1 wk, assessed the roles of each isoform on glucose metabolism and fiber growth. We achieved greater than 2.5-fold increases in total Ser473 phosphorylation in muscles expressing ca-Akt-1 and ca-Akt-2, respectively. Both isoforms caused hypertrophy of muscle fibers, consistent with increases in p70S6kinase phosphorylation, and a 60% increase in glycogen accumulation, although only Akt-1 increased glycogen synthase kinase-3beta phosphorylation. Akt-2, but not Akt-1, increased basal glucose uptake (by 33%, P = 0.004) and incorporation into glycogen and lipids, suggesting a specific effect on glucose transport. Consistent with this, short hairpin RNA-mediated silencing of Akt-2 caused reductions in glycogen storage and glucose uptake. Consistent with Akt-mediated insulin receptor substrate 1 (IRS-1) degradation, we observed approximately 30% reductions in IRS-1 protein in muscle overexpressing ca-Akt-1 or ca-Akt-2. Despite this, we observed no decrease in insulin-stimulated glucose uptake. Furthermore, a 68% reduction in IRS-1 levels induced using short hairpin RNAs targeting IRS-1 also did not affect glucose disposal after a glucose load. These data indicate distinct roles for Akt-1 and Akt-2 in muscle glucose metabolism and that moderate reductions in IRS-1 expression do not result in the development of insulin resistance in skeletal muscle in vivo.     

Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.     

RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.     

Short-term leptin-dependent inhibition of hepatic gluconeogenesis is mediated by insulin receptor substrate-2

Leptin has both insulin-like and insulin-antagonistic effects on glucose metabolism. To test whether leptin interferes directly with insulin signaling, we perfused isolated rat livers with leptin (0.1, 0.5, 5, and 25 nmol/liter), leptin + insulin (5 nmol/liter + 10 nmol/liter), insulin (10 nmol/liter), or vehicle (control). Leptin reduced L-lactate-(10 mmol/liter)-stimulated glucose production by 39-66% (P < 0.006 vs. control) and phosphoenolpyruvate carboxykinase (PEPCK) activity by 22-52% (P < 0.001). Physiological leptin concentrations (0.1-5 nmol/liter) stimulated the tyrosine phosphorylation (pY) of insulin receptor substrate-2 (IRS-2) (280-954%; P < 0.05) and its associated phosphatidylinositol-3 kinase activity (122-621%; P < 0.003). Leptin (0.5-25 nmol/liter) inhibited IRS-1 pY and its associated phosphatidylinositol-3 kinase activity (20-89%; P < 0.03) but stimulated janus kinase-2 pY (272-342%; P < 0.001). Leptin also down-regulated its short receptor isoform in a time- and concentration-dependent manner (28-54%; P < 0.05). Exposure to leptin + insulin additively reduced glucose production and PEPCK activity (approximately 50%; P < 0.001 vs. control) and doubled IRS-2 pY (P < 0.01 vs. insulin). However, leptin + insulin decreased IRS-1 pY by 57% (P < 0.01 vs. insulin). Insulin alone (P < 0.01), but not leptin, increased autophosphorylation of nonreceptor tyrosine kinases (pp59(Lyn) + pp125(Fak)). In conclusion, leptin both alone and in combination with insulin reduces hepatic glucose production by decreasing the synthesis of the key enzyme of gluconeogenesis, PEPCK, which results mainly from the stimulation of the IRS-2 pathway.     

Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC(50) for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t(1/2) of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.     

The direct involvement of SirT1 in insulin-induced insulin receptor substrate-2 tyrosine phosphorylation

NAD+ -dependent Sir2 family deacetylases and insulin signaling pathway are both conserved across species to regulate aging process. The interplay between these two genetic programs is investigated in this study. Protein deacetylase activity of SirT1, the mammalian homologue of Sir2, was suppressed through either nicotinamide treatment or RNA interference in several cell lines, and these cells displayed impaired insulin responses. Suppression of SirT1 activity also selectively inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2), whereas it had minimal effect on that of IRS-1. Further analyses showed that both IRS-1 and IRS-2 interacted with SirT1, and the acetylation level of IRS-2 was down-regulated by insulin treatment. Inhibition of SirT1 activity prevented deacetylation and insulin-induced tyrosine phosphorylation of IRS-2. Mutations of four lysine residues to alanine in IRS-2 protein, on the other hand, led to its reduced basal level acetylation and insulin-induced tyrosine phosphorylation. These results suggest a possible regulatory effect of SirT1 on insulin-induced tyrosine phosphorylation of IRS-2, a vital step in insulin signaling pathway, through deacetylation of IRS-2 protein. More importantly, this study may imply a pathway through which Sir2 family protein deacetylases and insulin signaling pathway jointly regulate various metabolic processes, including aging and diabetes.     

Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) inhibits insulin signal transduction in a variety of cell backgrounds, which might contribute to peripheral insulin resistance. However, because of the large number of potential phosphorylation sites, the mechanism of inhibition has been difficult to determine. One serine residue located near the phosphotyrosine-binding (PTB) domain in IRS-1 (Ser(307) in rat IRS-1 or Ser(312) in human IRS-1) is phosphorylated via several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK1. During a yeast tri-hybrid assay, phosphorylation of Ser(307) by JNK1 disrupted the interaction between the catalytic domain of the insulin receptor and the PTB domain of IRS-1. In 32D myeloid progenitor cells, phosphorylation of Ser(307) inhibited insulin stimulation of the phosphatidylinositol 3-kinase and MAPK cascades. These results suggest that inhibition of PTB domain function in IRS-1 by phosphorylation of Ser(307) (Ser(312) in human IRS-1) might be a general mechanism to regulate insulin signaling.