more about IRS1
Volume I
Morris F. White, Kyle D. Copps, in Endocrinology: Adult and Pediatric (Seventh Edition), 2016
Multisite Ser/Thr-Phosphorylation of IRS-Proteins
IRS1 and IRS2 can be regulated through a complex mechanism involving phosphorylation of more than 50 serine/threonine residues (phospho-S/Ts) within their unstructured tail regions.292Understanding how phospho-S/Ts regulate signaling is a difficult problem because so many sites and mechanisms appear to be involved. Heterologous signaling cascades initiated by proinflammatory cytokines or metabolic excess—including tumor necrosis factor-α (TNFα), endothelin-1, angiotensin II, excess nutrients (free fatty acids, amino acids, and glucose) or endoplasmic reticulum stress—are implicated in IRS1 phospho-S/T.293,294 Many biochemical and genetic experiments in cell-based systems suggest that individual phospho-S/T sites throughout the structure of IRS1 are associated with a reduction of insulin-stimulated tyrosine phosphorylation by up to 50%.295This level of inhibition is sufficient to cause glucose intolerance that could progress to diabetes, especially if pancreatic β-cells fail to provide adequate compensatory hyperinsulinemia.247
Numerous cell-based studies show IRS1 phospho-S/T to be a physiologically integrative mechanism modulating insulin sensitivity.292 Insulin is clearly an important input to IRS1phospho-S/T, as the vast majority of sites detected by specific monoclonal antibodies are stimulated by insulin, and they are diminished by the inhibition of the insulin-stimulated PI3K→Akt→mTOR cascade.296 Moreover, the IRS1 phospho-S/T patterns produced during drug-induced “metabolic stress” correlate significantly with that stimulated by insulin. These results suggest that IRS1 phospho-S/T is first and foremost a feedback mechanism that develops during insulin stimulation, but that this mechanism can be co-opted by metabolic stress—such as ER stress or inflammation—to inhibit insulin signaling and to promote metabolic disease.292,297-302 An implicit corollary is that hyperinsulinemia may be an important physiologic mediator of insulin resistance in animals, and there is some experimental evidence to corroborate this implication.303
In cultured cells, insulin-stimulated kinases—including aPKC (atypical protein kinase C), AKT, SIK2 (salt-inducible kinase 2), mTORC1, S6K1, ERL1/2 (extracellular signal-regulated kinase 1/2), ROCK1 (rho-associated coiled-coil containing protein kinase 1)—mediate feedback (autologous) IRS1 phospho-S/T with positive or negative effects on insulin sensitivity.292 An emerging view is that the positive/negative regulation of IRS1 by autologous pathways is subverted/co-opted in disease by inappropriate phospho-S/T levels mediated by heterologous kinases—including AMPK (AMP-activated protein kinase), GSK3 (glycogen synthase kinase 3), GRK2 (G protein-coupled receptor kinase 2), novel and conventional PKC isoforms, JNK (c-Jun N-terminal protein kinase), IKKβ (inhibitor of nuclear factor ĸB kinase β), and mPLK (mouse Pelle-like kinase). The use of siRNA shows additional kinases that might be involved, including Pim2 (Proviral Integrations of Moloney virus 2), PDHK (pyruvate dehydrogenase complex kinase), CaMKI-like, DAPK2 (death-associated protein kinase 2), DCLK1 (doublecortin-like kinase 1), STK10 (serine/threonine kinase 10), STK25 (serine/threonine kinase 25), MKK4 (MAP kinase kinase 4), MKK6 or MKK7, and LIMK2 (LIM domain kinase 2).304
One of the best-studied regulatory phosphorylation sites in IRS1is Ser307 (S307IRS1) in the rodent protein (human S312IRS1).301,305-309 Phosphorylation of S307IRS1 might be a common mechanism of insulin resistance (see Fig. 33-8). S307IRS1phosphorylation is associated with reduced tyrosine phosphorylation of IRS1 in cultured cells, which decreases the activation of the PI3K→AKT pathway in response to insulin.310,311Insulin itself promotes rat/mouse S307Irs1 phosphorylation through activation of the PI 3-kinase, showing feedback regulation that can be mediated by many kinases—PKCξ, IKKβ, JNK, mTOR, and S6K1 (see Fig. 33-8).295,296 Insulin-stimulated degradation of IRS1 via the PI3K pathway is in part dependent on the pS307IRS1.312 JNK can bind directly to IRS1, which appears to facilitate rat/mouse pS307Irs1 during stimulation of cells with proinflammatory cytokines. S307Irs1 is poorly phosphorylated in ob/ob (obese) mice that lack Jnk1, suggesting that this mechanism of inhibition has physiologic significance.313 Free fatty acids that contribute to insulin resistance promote pS307IRS1, possibly through PKCθ307; however, associated hyperinsulinemia has not been excluded. IRS1 can be phosphorylated by PKCδ on at least 18 sites in BL21 DE3 cells, including S307IRS1, S323IRS1, and S574IRS1, which appear to play an inhibitory role.314 Hyperactivated mTOR also promotes S307IRS1 phosphorylation, which is diminished in mice lacking S6K.288,315,316 IKKβ inhibitors (aspirin and salicylates) block S307IRS1 phosphorylation,306 which improves insulin sensitivity in obese rodents and in type 2 diabetes patients.317-319Phosphorylation of S307IRS1—located near the PTB domain—inhibits insulin-stimulated IRS1 tyrosine phosphorylation by disrupting the association between the insulin receptor and IRS1,311,320 although other phosphorylation sites might be involved.321
So far, only two studies have included direct investigations of the function of IRS1 S/T phosphorylation in mice using transgenesis or genetic knock-in to augment or replace endogenous (wild-type) IRS1 with a mutant version. Shulman and colleagues created transgenic mice having moderate overexpression in skeletal muscle (twofold vs. littermates) of nonmutant IRS1, or mutant IRS1 with alanine substitution of three serine residues—S302/307/612A (hS307/312/616A)—to block phosphorylation.322Thus, in the triple-mutant transgenic mice, possibly half the total IRS1 protein is endogenous in origin. Matching of IRS1expression is a potentially important consideration, given the role of autologous feedback. Regardless, the mutant transgenic mice showed better glucose tolerance than nonmutant transgenic mice when both were fed an HFD. Compared to an HFD with true wild-type littermates, the mutant transgenic mice showed increased total and muscle glucose disposal during clamp, and enhanced muscle IRS1 tyrosine phosphorylation and p85 binding in response to insulin. Although somewhat complicated in design, this experiment is consistent with the notion that S/T phosphorylation of IRS1 in skeletal muscle contributes to the development of insulin resistance in animals/humans.
We used genetic knock-in experiments to replace wild-type IRS1in mice with a mutant (A307IRS1) lacking the ability to be phosphorylated at S307 (equivalent to human S312IRS1).323 To control for potential ancillary effects of the knock-in process on IRS1 expression/function,324 nonmutant control knock-in mice were also generated (S307S, “S”). Surprisingly, given the sensitizing effect of the A307IRS1 mutation in cell-based assays, homozygous A307IRS1 mice show increased fasting insulin versus control mice, as well as very mild glucose intolerance. Furthermore, high-fat-fed mutant homozygous A307IRS1 mice exhibit higher fasting insulin and more severe glucose intolerance than wild-type mice, and the A307IRS1 protein exhibits decreased PI3K binding in insulin-stimulated primary hepatocytes. Thus, S307 phosphorylation appears permissive, rather than inhibitory, for insulin signaling in mice. But why is this? Among other, less prosaic explanations, it is possible that a serine is structurally required at position 307 of the IRS1 protein for its normal function. Alternatively, S307 phosphorylation could have a partial positive effect on insulin signal transduction that is more important in tissues/primary cells than its desensitizing function in continuous cell lines. By analogy with JNK1 activity, A307IRS1 phosphorylation could also have mixed, tissue-specific effects that are obscured by the standard knock-in approach.325In any case, the phenotype of A307IRS1 mice confirms that (non)phosphorylation of unique S/T sites on IRS1 can affect whole-body insulin sensitivity.
Growth Factor Regulation of Fetal Growth
Colin P. Hawkes, Lorraine E. Levitt Katz, in Fetal and Neonatal Physiology (Fifth Edition), 2017
Insulin Receptor Substrate and AKT Mutations
IRS-1 and IRS-2 mediate many insulin and IGF responses, especially those associated with somatic growth and carbohydrate metabolism. The IRS-1 branch of the pathway plays a significant role in mediating the effects of IGF-I on growth. Deletion of the Irs1 gene in mice reduces embryonic and neonatal growth,111 whereas deletion of Irs2 barely reduces prenatal and early postnatal growth, by 10%.112 The Irs1-knockout mouse has been described as having a phenotype of fetal growth restriction to 50% to 70% of normal size in the homozygous state.111 Postnatally, these mice remain small (at 50% to 70% of normal weight), and catch-up growth does not occur. The offspring display no obvious developmental abnormalities and have normal bone development. Gene targeting of Irs1 results in impairment of both growth and carbohydrate metabolism, compatible with its role in mediating both insulin and IGF-I effects.111,113 Growth is reduced by 40% in Irs2-null mice that are also haploinsufficient for Irs1, whereas growth is reduced 70% in Irs1-null mice also haploinsufficient for Irs2.114 Thus IRS-2 cannot fully replace IRS-1 in this process. Irs2-null mice also exhibit a diabetic phenotype with peripheral insulin resistance and pancreatic beta cell failure.112
Downstream from insulin receptor substrate, AKT1 and AKT2gene deletions also result in different phenotypes. Although Akt2-null mice exhibit insulin resistance,115 at birth Akt1-null mice have a 20% reduction in body weight compared with wild-type mice.116 The decrease in body weight in AKT-1-deficient mice persists throughout postnatal development regardless of sex, and glucose tolerance is normal.116 These data demonstrate that AKT-1 is more important to the growth of the organism, both in utero and after birth, whereas AKT-2 is critical to insulin-dependent control of carbohydrate metabolism.
IGF axis gene knockouts are summarized in Table 143-2.
IRS-1 and Vascular Complications in Diabetes Mellitus
I. Andrade Ferreira, J.W.N. Akkerman, in Vitamins & Hormones, 2005
VII
Summary and Future Perspectives
The discovery of IRS-1 occurred in 1991, and its characterization was the start of a massive research effort that marked the beginning of understanding the signaling events downstream of the IR and the tissue-specific behavior of insulin. IRS-1 is pivotal in the insulin signaling pathway, especially when mediating nonmitogenic stimuli. Mainly mitogenic stimuli downstream of IRS-1 signal back, thus forming an inhibitory feedback loop. It appears that in insulin resistance, this inhibition via mitogenic stimuli is preserved and that nonmitogenic stimulation mediated via IRS-1 is impaired (Fig. 6).
The dynamics of IRS-1 activation and degradation have been investigated primarily in adipocytes and muscle cells. Only 21st-century experiments have revealed a possible relation between abnormalities in IRS-1 regulation and the pathophysiological changes in blood and vessel walls related to thrombosis and atherosclerosis. Evidence suggests that insulin has the property to suppress inflammation, vascular tone, and the reactivity of platelets. This finding implies that a lack or defect in insulin-induced responses promotes inflammation, endothelial dysfunction, and platelet functions, thereby increasing the risk for atherothrombotic events and vascular occlusion. But much more work is required in this area.
In the future, patients with insulin resistance and DM will form one of the largest groups that will be treated in a hospital setting. Treatments will be focused on vascular complications associated with these disorders. More insight in the control of IRS-1 in blood cells and the vasculature will help the field of research understand the etiology of vascular complications in these patients and make room for the development of better treatment regimens.
G Protein-Coupled Receptors in Energy Homeostasis and Obesity Pathogenesis
Ziru. Li*†, ... Weizhen. Zhang*†‡, in Progress in Molecular Biology and Translational Science, 2013
1.9.4
PI3K/AKT signaling
Insulin receptor substrate (IRS-1)-associated PI3K activity and Akt phosphorylation are also regulated by ghrelin. In hepatoma cells, ghrelin upregulates IRS-1-associated PI3K activity.88 On the other hand, ghrelin inhibits Akt kinase activity and partially reverses the downregulation of insulin on phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression, a rate-limiting enzyme of gluconeogenesis that catalyzes the conversion of oxaloacetate to phosphoenolpyruvate.
Ghrelin also stimulates the IRS-1-associated PI3K/Akt pathway in 3T3-L1 preadipocytes, whereas inhibition of PI3K blocks the effects of ghrelin on the proliferation and apoptosis of these cells. Furthermore, ghrelin increases basal and insulin-stimulated glucose transport. The effect of ghrelin on glucose transport can be blocked by coadministration of a PI3K inhibitor.85 These findings indicate that the PI3K/Akt pathway acts to mediate the effects of ghrelin on 3T3-L1 cells.
Annual Reports in Medicinal Chemistry
Philip A. Carpino, David Hepworth, in Annual Reports in Medicinal Chemistry, 2012
2.1.1
PTP1b inhibitors
PTB1b negatively regulates insulin signaling by catalyzing the dephosphorylation of both the IR and insulin-receptor substrate-1 (IRS1). Genetic polymorphism data associate PTP1b with protection from and/or development of insulin resistance and diabetes in humans.20 Ablation of PTP1b in mice improves insulin sensitivity and prevents weight gain on a high fat diet.21
The identification of small molecule PTP1b inhibitors with appropriate pharmacokinetic properties and selectivity has proven extremely challenging.22 Many competitive PTP1b inhibitors are phosphotyrosine mimetics that contain a carboxylic or phosphonic acid (i.e., to drive binding affinity to the high-affinity catalytic site) and a large lipophilic tail seeking to maximize the additional binding energy that can be obtained from the shallow binding pocket.23 These PTP1b inhibitors generally exhibit low ligand efficiency, poor cell activity, and suboptimal pharmacokinetic properties. Application of acidic isosteres has led to some improvement in ADME properties, but not sufficient to produce clinical candidates against the target.24An allosteric binding site on the enzyme has been identified but not yet successfully exploited in the design of oral inhibitors.25The discovery of new PTP1b inhibitors remains an active area of research, especially in academia, but further breakthroughs will be required to deliver orally active agents.26
While PTP1b does not appear to be druggable from a small molecule perspective, PTP1b protein levels can be modulated using antisense oligonucleotides (ASOs) that bind mRNA and reduce protein transcription. A PTP1b ASO ISIS113715 was advanced into Phase II clinical trials and shown to reduce plasma glucose and LDL-cholesterol in diabetic patients without causing weight gain.27 Development of ASO ISIS113715 has been reportedly suspended, replaced by a new ASO ISIS-PTP1BRX(structure unknown), currently in Phase I trials.28
Role of mTOR Signaling in Cardioprotection
Anindita Das, Rakesh C. Kukreja, in Molecules to Medicine with mTOR, 2016
15.7
mTOR Signaling in Adipogenesis and Lipogenesis
mTORC1 activity is high in the livers of obese rodents, which leads to degradation of IRS1 and poor insulin signaling, as well as hepatic insulin resistance [109]. Lipogenesis is paradoxically very active in the liver of insulin-resistant rodents [24]. mTORC1 hyperactivation by overfeeding promotes lipogenesis through induction of SREBP-1c (SREBP) cleavage and activation [110,111]. Insulin activates mTORC1 through Akt-mediated phosphorylation and inhibition of TSC2 (tuberous sclerosis complex 2) [39–41]. Rapamycin blocks Akt-induced SREBP-1 expression and nuclear accumulation, the expression of several lipogenic genes, and the synthesis of various classes of lipids [111]. The knockdown of Raptor, but not Rictor, showed similar effects, indicating that SREBP-1 activation mainly depends on mTORC1, but not mTORC2 [28,112,113]. Using the mTORC1 inhibitor, rapamycin, other independent studies also confirmed the significant role of mTORC1 in the regulation of energy production through profound effects on hepatic fatty acid metabolism [114,115]. Additionally, the antidiabetic drug, metformin, which is a known to activate adenosine monophosphate-activated protein kinase (AMPK) and also subsequently inhibits mTORC1, reduced hepatic lipid content by promoting fatty acid oxidation, impairing SREBP-1c expression and cleavage [116]. In addition, using liver-specific Rictor knockout mice, a recent study has established the crucial role of hepatic mTORC2 in lipogenesis through activation of Akt-mTORC1-SREBP-1c [117].
Pharmacological and genetic studies have demonstrated that mTORC1 signaling also plays a fundamental role in lipid storage by stimulating the synthesis of triglycerides in white adipose tissue (WAT) and the differentiation of preadipocytes into white adipocytes through the translational control of the master regulator of adipogenesis, PPAR-γ [118,119]. mTOR inhibition with rapamycin reduced mRNA and protein levels of PPAR-γ and C/EBP-α (CCAAT/enhancer binding protein) and the expression of numerous lipogenic genes [118,120,121]. Constitutive activation of mTORC1 through TSC2 deletion or the deletion of 4E-BP1/2 induces PPAR-γ and C/EBP-α expression and promotes adipogenesis [102,119]. Additionally, S6K1-deficient mice had reduced adipose tissue mass and were protected against diet-induced obesity [106]. Adipocyte-specific deletion of Raptor in mice resulted in lean mice with reduced WAT mass which are resistant to high-fat diet (HFD)-induced obesity [113].
Volume I
David R. Clemmons, in Endocrinology: Adult and Pediatric (Seventh Edition), 2016
Receptor-Mediated Signal Transduction
Following activation of the intrinsic tyrosine kinase activity and phosphorylation of tyrosine 950, the docking protein, insulin receptor substrate-1 (IRS-1), binds directly to the receptor through its PTB domain (Fig. 21-4). A functionally similar protein termed IRS-2 has been shown to bind by a similar mechanism. Following binding, IRS-1 is tyrosine phosphorylated by the receptor at multiple sites, creating docking motifs that provide docking sites for intracellular signaling proteins that contain Src homology-2 (SH-2) domains. These domains contain approximately 100 amino acids that share sequence similarity cellular oncogene, Src. Several tyrosines in IRS-1 occur within sequences that provide a recognition motif for SH-2 domains. IRS-1 gene deletion in mice results in a major decrease in body weight with proportionate reduction in liver, heart, and spleen.33Activation of signaling pathways that lead to enhanced IRS-1 degradation result in attenuation of IGF-1 signaling. Serine phosphorylation of IRS-1 can lead to its recognition by chaperones that mediate its translocation to proteasomes and ultimately its degradation. This occurs in response to hyperglycemic stress. Additionally, serine phosphorylation can function to directly inhibit IGF-1–stimulated tyrosine phosphorylation. For example, during energy restriction AMP kinase is activated and phosphorylates IRS-1 Ser794, which inhibits IGF-1 receptor–stimulated tyrosine phosphorylation, thereby inhibiting the ability of IGF-1 to stimulate protein synthesis.
Signaling proteins that bind directly to the phosphorylated tyrosines on IRS-1 include the adaptor proteins, Shc and Grb-2, and the p85 subunit of PI-3 kinase. Grb-2 forms a complex with the Ras-activating protein (Son of Sevenless [SOS]), and this complex leads to subsequent P-21 Ras activation, which activates Raf and downstream components of the MAP kinase pathway. Activation of this pathway is important for the mitogenic function of IGF-1.
IRS-1 phosphorylation also results in binding of the p85 subunit, and this leads to recruitment of the catalytic subunit, p110, and its activation.34 This results in generation of inositol triphosphate and activation of protein tyrosine kinase B. This kinase activates mTOR and P70 S6 kinase, which leads to activation of protein translation. This pathway is utilized by IGF-1 to stimulate protein synthesis, and it mediates its major anabolic actions. This pathway is also important for IGF-1–induced increases in cell motility and for inhibition of apoptosis. AKT also phosphorylates GSK-3 beta, leading to its inactivation, which is important for several responses including stimulation of glucose transport.
Sustained levels of activated IRS-1 also lead to IGF-1–stimulated differentiation, and this response is mediated through PI-3 kinase/AKT activation. Activation of this pathway has been shown to be important for skeletal muscle and chondrocyte, osteoblast differentiation.35,36 Stimulation of the MAP kinase, which is important for cell proliferation, requires Grb-2/Shc association with IRS-1. It may also require localization of IRS-1 and MAP kinase within specific subcellular locations.37 Pathophysiologic conditions such as oxidative stress,38 glucocorticoid excess,39hyperglycemia40 and cytokine activation41 result in IRS-1 downregulation. In some cell types, such as skeletal muscle and cartilage, this results in atrophy39 or apoptosis.38 Other cell types, such as vascular endothelium or smooth muscle, respond to IRS-1 downregulation by activating aberrant signaling pathways that are activated in response to IGF-1. Although these cell types do not undergo atrophy or apoptosis, aberrant pathway activation can result in pathophysiologic changes in cellular functions.40 The IGF-1 receptor can directly bind and phosphorylate p52Shc, and this leads to Shc association with Grb-2, which activates RAS and MAPK independently of IRS-1. In contrast, in some pathophysiologic situations as increased oxidative stress, Shc activation proceeds by a different mechanism. In vascular cell types that express the αVβ3 integrin, oxidative stress leads to increased secretion of αVβ3 ligands. αVβ3 activation results in translocation of an adaptor protein SHP-2 to the plasma membrane–associated scaffolding protein SHPS-1.42 The IGF-1 receptor phosphorylates SHPS-1, which results in recruitment of Src and the NAPDH oxidase, Nox4. Nox4 activates Src, which phosphorylates Shc.43 This IRS-1–independent MAP kinase leads to smooth-muscle cell proliferation or endothelial dysfunction.
An important mediator of IGF-1 action is the nuclear protein FOXOA1. This protein is serine phosphorylated in response to AKT activation, which leads to its transport out of the nucleus.44This results in decreased expression of proteins that regulate apoptosis and the cell stress response. The IGF-1 receptor also signals through direct interactions with G proteins.45 Similarly, activation of protein kinase C isoforms has been shown to alter signaling through IGF-1R.43 Several signaling intermediates have been shown to modulate or directly inhibit IGF-1R signaling. One of the best examples is the SOCS proteins, which also inhibit GH receptor signaling. Cytokines such as Il-1 and IL-6 that antagonize IGF-1 actions activate SOCS3, which inhibits IRS-1 activation.46 The sirtuins, which are histone deacetylases, also negatively modulate IGF-1–stimulated AKT activation.47
Specific intracellular signaling pathways have been shown to mediate specific IGF-1 actions. The PI3-kinase pathway is important for glucose transport, cell migration differentiation, protein synthesis, and hypertrophy, and inhibitors of PI 3-kinase have been shown to inhibit these IGF-1–stimulated effects.34,38,40Similarly, the MAP kinase pathway is the predominant pathway for mitogenesis and rescue from apoptosis.38,48 Protein kinase C isoforms facilitate IGF-1–stimulated cell migration and stimulation of the transcription of specific genes. The induction of specific proteins by IGF-1 has been shown to be required for proliferation,49,50 differentiation,51,52 and protein synthesis and apoptosis.53 However, the requirement of stimulation of a specific pathway for a specific function is not absolute, since the results generated using specific inhibitors of each pathway support the conclusion that there are overlapping functions.
In addition to interactions between the IGF-1 and insulin receptor–linked signaling pathways, several other signaling pathways have been shown to influence IGF-1–stimulated signaling events. Several hormones and growth factors such as EGF, angiotensin 2, aldosterone, endothelin-1, PTH, PTHrP, and sonic hedgehog have been shown to activate signaling intermediates that modulate IGF-1 receptor–linked signaling events.54-57 Conversely, cellular activation by IGF-1 has been shown to result in transactivation of the androgen receptor, EGFR, VEGFR, and the chemokine receptor CXR4.58,59 In addition, postreceptor signaling pathway cross-talk has been demonstrated for the GH, estrogen, androgen, progesterone, and glucocorticoid receptors.60-62
Volume I
John J. Kopchick, ... Lawrence A. Frohman, in Endocrinology: Adult and Pediatric (Seventh Edition), 2016
Insulin Receptor Substrate/PI3K-AKT Signaling Pathway
In addition to sharing some MAPK pathway intermediates with insulin, GH activates members of an additional insulin signaling pathway: IRS-1 and IRS-2. Although the nature of the interaction between the IRS molecules and the GHR/JAK2 complex is not clear, it does appear that JAK2 activation results in tyrosyl phosphorylation of IRS-1 and IRS-2, which is involved in the insulin-like effects of GH. Phosphoinositol-3-kinase (PI3K) is also involved in the insulin-like effects of GH, in that a GH-induced interaction between the regulatory subunit of PI3K and tyrosyl phosphorylated IRS-1 and IRS-2 has been demonstrated in adipocytes. The ability of the PI3K-AKT pathway to promote cell proliferation and differentiation and to prevent apoptosis has been well documented.
A role for GH induction of the PI3K-AKT pathway in GH regulation of IGF-1 expression cannot be ruled out, because inhibition of this pathway results in reduction of GH-induced IGF-1 expression in mouse cells. Nevertheless, targeted gene disruption studies of the p85α regulatory subunit of PI3K, as well as the downstream effector of PI3K-AKT, indicate that, although the PI3K-AKT pathway is essential for survival and normal growth, its effects are not necessarily direct functions of GH action.99
Volume 3
Morris F. White, in Handbook of Cell Signaling (Second Edition), 2010
Insulin Receptor Substrates
Cell based and mouse based experiments show that most if not all insulin signals are produced or modulated through tyrosine phosphorylation of IRS1, IRS2, or its homologs; or other scaffold proteins including SHC, CBL, APS and SH2B, GAB1, GAB2, DOCK1, and DOCK2 [19–25]. Although the role of each of these substrates merits attention, work with transgenic mice suggests that many insulin responses – especially those that are associated with somatic growth and nutrient homeostasis – are mediated through IRS1 or IRS2 [26].
The IRS-proteins are adapter molecules that link the insulin-like receptors to common downstream signaling cascades (Figure 331.1). Four Irs-protein genes have been identified in rodents, but only three of these genes (IRS1, IRS2, and IRS4) are expressed in humans [27]. IRS1 and IRS2 are broadly expressed in mammalian tissues, whereas IRS4 is largely restricted to the hypothalamus [28]. Each of these proteins is targeted to the activated insulin-like receptors through an NH2-terminal pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain (Figure 331.2a). The PTB domain binds specifically to the phosphorylated NPEY motif in the activated receptor kinases [29]. This interaction is important, as deletion of the PTB domain or deletion of Tyr960/972 in the IR impairs signal transduction [17, 30]. The PH domain also promotes the interaction between IRS-proteins and the IR, but the mechanism is poorly understood. Although PH domains are generally thought to bind phospholipids, the PH domains in IRS1 and IRS2 are poor examples of this specificity [31, 32]. Regardless, the PH domain in the IRS-protein plays an important and specific role as it can be interchanged among the IRS-proteins without noticeable loss of bioactivity, whereas heterologous PH domains inhibit IRS1 function when substituted for the normal PH domain [33].
In addition to the PH and PTB domains, IRS2 utilizes an additional mechanism to interact with the activated insulin receptor [34]. This IRS2 specific interaction was originally localized between amino acid residues 591 and 786 in IRS2, and subsequently shown to require Tyr624 and Tyr628 in IRS2 [35]. This binding region in IRS2 was originally called the kinase regulatory loop binding (KRLB) domain because the interaction was shown to require the three tyrosine autophosphorylation sites in the regulatory loop of the functional receptor kinase [34]. However, Wu et al. showed recently that receptor autophosphorylation was required to move the regulatory loop out of the catalytic site so the functional part of the KRLB domain residues 620–634 in murine IRS2 can bind in the catalytic site with Tyr621 inserted into the ATP binding pocket and Tyr628 positioned for phosphorylation [36] (Figure 331.2b). Tyr628might be a critical phosphorylation site that needs to be oriented properly by the KRLB domain for immediate phosphorylation upon insulin binding. Alternatively, the KRLB domain might attenuate signaling through IRS2 by blocking ATP access to the catalytic site. Further work is needed to establish the physiological significance of this unique motif in IRS2.
IRS→PI3K→AKT Cascade
One of the best studied insulin-like signaling cascades involves the production of PI-3,4,5-P3 by the phosphatidylinositol 3-kinase (PI 3-kinase). The type 1 PI 3-kinase is composed of a regulatory subunit that contains two src-homology-2 (SH2) domains and a catalytic subunit that is inhibited by the regulatory subunit until its SH2 domains are occupied by phosphorylated tyrosine residues in the IRS-proteins [37]. PI-3,4,5-P3 recruits the Ser/Thr kinases PDK1 and AKT (also known as PKB) to the plasma membrane where AKT is activated by PDK1 mediated phosphorylation (Figure 331.3). AKT phosphorylates many proteins that play a central role in cell survival, growth, proliferation, angiogenesis, metabolism, and migration [38]. Phosphorylation of several genuine AKT substrates is especially relevant to insulin-like signaling: GSK3α/β (blocks inhibition of glycogen synthesis), AS160 (promotes GLUT4 translocation), the BAD•BCL2 heterodimer (inhibits apoptosis), the FOXO transcription factors (regulates gene expression), p21CIP1 and p27KIP1 (blocks cell cycle inhibition), eNOS (stimulates NO synthesis and vasodilatation), and PDE3b (hydrolyzes cAMP) (Figure 331.3). AKT also phosphorylates tuberin (TSC2), which inhibits its GAP activity toward the small G-protein RHEB promoting the accumulation of the RHEB•GTP complex that activates mTOR [38, 39]: This pathway provides a direct link between insulin signaling and protein synthesis that is needed for cell growth (Figure 331.3).
The role of IRS-proteins in the PI3K→AKT signaling cascade is validated by a wide array of cell based and mouse based experiments. Although IRS1 was originally purified and cloned from rat hepatocytes, the principle role of Irs1 and Irs2 during insulin signaling in hepatocytes in vivo was verified only recently [40–43]. The simplest experiments employ an intraperitoneal injection of insulin into ordinary mice, or mice lacking hepatic IRS1, IRS2, or both substrates. In ordinary mice, insulin rapidly stimulates Akt phosphorylation, and the phosphorylation of its downstream substrates Foxo1 and Gsk3α/β. However, both IRS1and IRS2 must be deleted before insulin receptors are uncoupled from the PI3K→AKT cascade [42]. These results confirm the shared but absolute requirement for IRS1 or IRS2 for the hepatic insulin signaling.
The IRS→PI3K→AKT cascade is very robust owing to the expression of multiple isoforms of each component that confers both redundancy and specificity upon the system [8]. For example, the type 1A PI 3-kinase is a dimer assembled from one catalytic subunit, p110α or p110β, and one of eight regulatory subunit isoforms encoded by three different genes, Pik3r1, Pik3r2, and Pik3r3 [38]. In most cells including the liver, p85α and its two shorter isoforms p55α and p50α encoded by Pik3r1 constitute most of the regulatory subunits; however, p85β encoded by Pik3r2 also contributes to the total pool of regulatory subunits [44, 45]. Thus, both genes must be inactivated to reduce the cellular concentration of PIP3 by more than 90 percent and confirm the role of PI3K in the hepatic insulin signaling cascade [46].
AKT is also encoded by thee genes. However, mouse based experiments clearly show that these isoforms are not redundant components of the insulin-like signaling cascade. AKT1 deficient mice display developmental differences and are generally smaller than littermate controls [47]. By comparison, AKT2 deficient mice display metabolic defects; and AKT3 deficient mice display neural defects [38]. AKT2 rather than AKT1 is primarily responsible for insulin stimulated GLUT4 translocation in adipose tissues [48]. More work is needed to understand the beneficial and detrimental effects of the redundant signaling, how AKT isoforms generate selective metabolic signals, and whether this diversity can be exploited to treat metabolic disease and cancer.
Volume 2
Mark A. Lemmon, in Handbook of Cell Signaling (Second Edition), 2010
Possible Roles of Non-Phosphoinositide PH Ligands
PH domains for which protein targets have been reported include both examples that bind phosphoinositides weakly and promiscuously (e.g., the βARK, IRS-1, and dynamin PH domains), as well as PH domains that bind strongly and specifically to particular phosphoinositides (e.g., the Btk and PKB PH domains). It can therefore not be argued that the protein targets described for PH domains are simply alternatives to, or surrogates for, the well-studied (but rare) specific phosphoinositide ligands. Rather, it appears likely that some PH domains bind multiple ligands.
Cooperation of Multiple Ligands in Membrane Recruitment of PH Domains
A requirement for simultaneous PH domain binding to two different ligands was first demonstrated for membrane targeting by the βARK PH domain [83]. The βARK PH domain binds very weakly to PtdIns(4,5)P2 (KD>200 μM) [12]. It also binds rather weakly to the βγ-subunits of heterotrimeric G-proteins [82]. Neither of these weak interactions alone is sufficient for high-affinity targeting of βARK to membranes, but the two interactions can cooperate to recruit βARK efficiently to relevant membrane surfaces [83].
Golgi Targeting of PH Domains by Multiple Ligands
The PH domain from oxysterol binding protein (OSBP), as well as several other related PH domains, is targeted specifically to the Golgi through interactions that appear to require both phosphoinositides and another unidentified (Golgi-specific) component [96]. These Golgi-targeted PH domains, which include those from FAPP1 and the Goodpasture antigen binding protein (GPBP), are highly promiscuous in their phosphoinositide binding (and are not PtdIns-4-P-specific) [34, 96], arguing that phosphoinositide recognition alone cannot possibly determine their Golgi targeting. Phosphoinositide binding by these PH domains is several-fold weaker than PtdIns(4,5)P2 binding by the PLC-δ1 PH domain ([96], and D. Keleti, V. J. Sankaran, and M. A. Lemmon, unpublished), further suggesting that it may not be strong enough to drive membrane targeting of the OSBP PH domain independently. Studies in a series of yeast mutants have demonstrated that Golgi targeting of the OSBP and FAPP1 PH domains is dependent on PtdIns-4-P and not on PtdIns(4,5)P2 production [96, 97], but that the activity of Arf1p is also important [96]. It is therefore hypothesized that the presence of two binding partners in the Golgi is responsible for specific targeting of the OSBP, FAPP1, and GPBP PH domains to that organelle. On its own, phosphoinositide binding by these PH domains is not strong enough to drive membrane targeting in vivo, and would certainly not provide targeting specificity. The second (so far unidentified) target of these PH domains is thought to be Golgi-specific, but does not bind to the PH domains tightly enough to achieve Golgi targeting on its alone. Rather both phosphoinositide and this unknown component must be present in the same membrane (the Golgi) in order to recruit the OSBP and other PH domains to that compartment with high affinity and specificity. According to this model [96], PtdIns-4-P is implicated in Golgi targeting of the OSBP, FAPP1, and other PH domains not because of headgroup recognition, but because this happens to be the most abundant phosphoinositide in the membranes that contain the second PH domain ligand.
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