On the role of IRS2 in the regulation of functional beta-cell mass

The proper regulation of blood glucose homeostasis in mammals requires an adequate relation between the capacity to produce insulin and metabolic demand. Insulin receptor substrate proteins (IRS) are signalling intermediates that are required to keep this balance because they are needed for insulin action in target tissues but also for insulin production in pancreatic beta-cells. The total functional beta-cell mass in an individual sets the limit of how much insulin can be produced at a given time. It can change adaptively to meet demand and studies in vivo indicate that the regulation of beta-cell mass involves IRS2, while IRS1 is only required for proper insulin production in beta-cells. Overexpression studies in isolated islets have shown that IRS2, but not IRS1 or Shc, is sufficient to induce proliferation of beta-cells and to protect against d-glucose-induced apoptosis. In light of the finding that many growth factors can regulate Irs2 in islets, this signalling intermediate could balance capacity for insulin production with demand. This review summarizes observations in mouse models and in primary beta-cells and proposes a new hypothetical model of how IRS2 might control beta-cell mass.     

Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes

Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO(2) at 37 degrees C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.     

Insulin signalling: the role of insulin receptor substrate 1

The insulin receptor is a ligand-activated tyrosine kinase that phosphorylates its major substrate protein, insulin receptor substrate 1 (IRS1), at multiple sites. Tyrosine-phosphorylated IRS1 then serves as a docking/effector protein for at least four Src homology 2 (SH2)-domain proteins involved in signal transduction. This initial step in signalling distinguishes the insulin receptor from other receptor tyrosine kinases, which directly bind several SH2-domain proteins, and establishes IRS1 as a founding member of a group of proteins whose function is to link activated tyrosine kinases to SH2-domain proteins.     

Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action

Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1^−/− and IRS-2^−/− mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins.     

IRS2 and PTP1B: Two opposite modulators of hepatic insulin signalling

Type 2 Diabetes mellitus (T2D) is the most common endocrine disorder associated to metabolic syndrome (MS) and occurs when insulin secretion can no compensate peripheral insulin resistance. Among peripheral tissues, the liver controls glucose homeostasis due to its ability to consume and produce glucose. The molecular mechanism underlying hepatic insulin resistance is not completely understood; however, it involves the impairment of the insulin signalling network. Among the critical nodes of hepatic insulin signalling, insulin receptor substrate 2 (IRS2) and protein tyrosine phosphatase 1B (PTP1B) modulate the phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 pathway that controls the suppression of gluconeogenic genes. In this review, we will focus on recent findings regarding the molecular mechanism by which IRS2 and PTP1B elicit opposite effects on carbohydrate metabolism in the liver in response to insulin. Finally, we will discuss the involvement of the critical nodes of insulin signalling in non-alcoholic fatty liver disease (NAFLD) in humans.     

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

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

SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.     

SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2

Inflammation associates with peripheral insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. Inflammation induces the expression of SOCS proteins. We show that SOCS1 or SOCS3 targeted IRS1 and IRS2, two critical signaling molecules for insulin action, for ubiquitin-mediated degradation. SOCS1 or SOCS3 bound both recombinant and endogenous IRS1 and IRS2 and promoted their ubiquitination and subsequent degradation in multiple cell types. Mutations in the conserved SOCS box of SOCS1 abrogated its interaction with the elongin BC ubiquitin-ligase complex without affecting its binding to IRS1 or IRS2. The SOCS1 mutants also failed to promote the ubiquitination and degradation of either IRS1 or IRS2. Adenoviral-mediated expression of SOCS1 in mouse liver dramatically reduced hepatic IRS1 and IRS2 protein levels and caused glucose intolerance; by contrast, expression of the SOCS1 mutants had no effect. Thus, SOCS-mediated degradation of IRS proteins, presumably via the elongin BC ubiquitin-ligase, might be a general mechanism of inflammation-induced insulin resistance, providing a target for therapy.     

Role of IRS and PHIP on insulin-induced tyrosine phosphorylation and distribution of IRS proteins

To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.     

Phosphorylation of IRS proteins, insulin action, and insulin resistance

Insulin signaling at target tissues is essential for growth and development and for normal homeostasis of glucose, fat, and protein metabolism. Control over this process is therefore tightly regulated. It can be achieved by a negative feedback control mechanism whereby downstream components inhibit upstream elements along the insulin-signaling pathway (autoregulation) or by signals from apparently unrelated pathways that inhibit insulin signaling thus leading to insulin resistance. Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues has emerged as a key step in these control processes under both physiological and pathological conditions. The list of IRS kinases implicated in the development of insulin resistance is growing rapidly, concomitant with the list of potential Ser/Thr phosphorylation sites in IRS proteins. Here, we review a range of conditions that activate IRS kinases to phosphorylate IRS proteins on "hot spot" domains. The flexibility vs. specificity features of this reaction is discussed and its characteristic as an "array" phosphorylation is suggested. Finally, its implications on insulin signaling, insulin resistance and type 2 diabetes, an emerging epidemic of the 21st century are outlined.     

IRS proteins and the common path to diabetes

Although a full understanding of insulin/insulin-like growth factor (IGF) action is evolving, the discovery of insulin receptor substrate (IRS) proteins and their role to link cell surface receptors to the intracellular signaling cascades provided an important step forward. Moreover, Insulin/IGF receptors use common signaling pathways to accomplish many tasks, the IRS proteins add a unique layer of specificity and control. Importantly, the IRS-2 branch of the insulin/IGF-signaling pathway is a common element in peripheral insulin response and pancreatic beta-cell growth and function. Failure of IRS-2 signaling might explain the eventual loss of compensatory hyperinsulinemia during prolonged periods of peripheral insulin resistance. Moreover, short-term inhibition of IRS protein functions by serine phosphorylation, or sustained inhibition by ubiquitin-targeted proteosome-mediated degradation suggests a common molecular mechanism for insulin resistance during acute injury or infection, or the sensitivity of beta-cells to autoimmune destruction. The broad role of IRS-1 and IRS-2 in cell growth and survival reveals a common regulatory pathway linking development, somatic growth, fertility, neuronal proliferation, and aging to the core mechanisms used by vertebrates for nutrient sensing.     

Molecular mechanisms of insulin receptor substrate protein-mediated modulation of insulin signalling

The insulin receptor substrate (IRS) proteins act as important mediators of insulin action. Their regulation serves to augment the specificity of the insulin signalling cascade. They can be regulated--both positively and negatively--at the level of phosphorylation, and signalling through these proteins can be further modulated through the actions of SOCS (suppressor of cytokine signalling) proteins. Understanding the mechanisms of IRS regulation will provide further insight into the pathophysiology of insulin resistance and type 2 diabetes.     

The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling.

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein--protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules.     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

Two new substrates in insulin signaling,IRS5/DOK4 and IRS6/DOK5

We have identified two new human genes that encode proteins with tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at their amino termini. Because the other known PH-PTB proteins (insulin receptor substrates: IRS-1, IRS-2, IRS-3, and IRS-4, and the downstream of kinases: DOK-1, DOK-2, and DOK-3) are substrates of insulin and insulin-like growth factor (IGF)-1 receptors, we asked whether these new proteins, termed IRS5/DOK4 and IRS6/DOK5, might also have roles in insulin and IGF-1 signaling. Northern analyses indicate that IRS5/DOK4 is ubiquitously expressed but most abundant in kidney and liver. IRS6/DOK5 expression is highest in skeletal muscle. Both proteins are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics differ. Insulin receptor-phosphorylated IRS5/DOK4 associates with RasGAP, Crk, Src, and Fyn, but not phosphatidylinositol 3-kinase p85, Grb2, SHP-2, Nck, or phospholipase Cgamma Src homology 2 domains, and activates MAPK in cells. IRS6/DOK5 neither associates with these Src homology 2 domains nor activates MAPK. IRS5/DOK4 and IRS6/DOK5 represent two new signaling proteins with potential roles in insulin and IGF-1 action.     

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

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

Nexilin,a Cardiomyopathy-Associated F-Actin Binding Protein,Binds and Regulates IRS1 Signaling in Skeletal Muscle Cells

Insulin stimulates glucose uptake through a highly organized and complex process that involves movement of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Previous studies in L6 skeletal muscle cells have shown that insulin-induced activation and assembly of insulin receptor substrate 1 (IRS1) and p85α the regulatory subunit of the Type 1A phosphatidylinositol-3-kinase (PI3K), within remodeled actin-rich membrane structures is critical for downstream signalling mediating the translocation of GLUT4. The mechanism for localization within actin cytoskeletal scaffolds is not known, as direct interaction of IRS1 or p85α with F-actin has not been demonstrated. Here we show that nexilin, a F-actin binding protein implicated in the pathogenesis of familial dilated cardiomyopathies, preferentially binds to IRS1 over IRS2 to influence glucose transport in skeletal muscle cells. Nexilin stably associates with IRS1 under basal conditions in L6 myotubes and this complex is disassembled by insulin. Exposure of L6 myotubes to Latrunculin B disrupts the spatial patterning of nexilin and its transient association with IRS1. Functional silencing of nexilin has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, however it enhances recruitment of p85α to IRS1 resulting in increased PI-3, 4, 5-P₃ formation, coincident with enhanced AKT activation and glucose uptake. By contrast, overexpression of nexilin inhibits transmission of IRS1 signals to AKT. Based on these findings we propose that nexilin may tether IRS1 to actin-rich structures under basal conditions, confining IRS1 signaling to specific subcellular locations in the cell. Insulin-elicited release of this constraint may enhance the efficiency of IRS1/PI3K interaction and PI-3, 4, 5-P₃ production at localized sites. Moreover, the selective binding of nexilin to IRS1 and not IRS2 may contribute to the differential specificity of IRS isoforms in the modulation of GLUT4 trafficking in skeletal muscle cells.     

Expression of insulin signalling components in the sensory epithelium of the human saccule

Several studies have demonstrated a link between diabetes and the dysfunction of the inner ear. Few studies, however, have reported the signalling mechanisms involved in metabolic control in human inner ear cells. Knowledge of the expression and role of the insulin receptor and downstream signalling components in the inner ear is sparce. Our immunohistochemistry approach has shown that the insulin receptor, insulin receptor substrate 1 (IRS1), protein kinase B (PKB) and insulin-sensitive glucose transporter (GLUT4) are expressed in the sensory epithelium of the human saccule, which also exhibits expression of a calcium-sensitive cAMP/cGMP phosphodiesterase 1C (PDE1C) and the vasopressin type 2 receptor. IRS1 and PDE1C are selectively expressed in sensory epithelial hair cells, whereas the other components are expressed in sensory epithelial supporting cells or in both cell types, as judged from co-expression or non-co-expression with glial fibrillary acidic protein, a marker for supporting cells. Furthermore, IRS1 appears to be localized in association with sensory nerves, whereas GLUT4 is expressed in the peri-nuclear area of stromal cells, as is the case for aquaporin 2. Thus, the insulin receptor, insulin signalling components and selected cAMP signalling components are expressed in the human saccule. In addition to well-known mechanisms of diabetes complications, such as neuropathy and vascular lesions, the expression of these proteins in the saccule could have a role in the observed link between diabetes and balance/hearing disorders.     

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.     

Mapping the Structural Topology of IRS Family Cascades Through Computational Biology

Structural topologies of proteins play significant roles in analyzing their biological functions. Converting the amino acid data in a protein sequence into structural information to outline the function of a protein is a major challenge in post-genome research which can add an extra room in understanding the protein sequence–structure–function relationships. In this study, we performed a comprehensive bioinformatics analysis of structural topology of the IRS family members such as IRS-1, IRS-2, IRS-3, IRS-4, IRS-5 and IRS-6. Based on this assessment, we found that IRS-2 encloses the highest number of α helices, β sheets and β turns in the secondary structure topology compared to IRS-1 and IRS-6. IRS family members are rich in serine or leucine residues. Among the IRS family members, the highest percentage of serine and leucine was observed in IRS-1 (15 %) and IRS-5 (10 %), respectively. Notably, the highest number of disulphide bonds was observed in IRS-1 (10) which is responsible for structural stability of the protein. Hydrogen bond pattern in α helices and β sheet was recorded in IRS-1, IRS-2 and IRS-6. By conservation analysis, the longest protein IRS-3 was found to be highly conserved among the IRS family members. The cluster of sequence logo present in the N terminus of these cascades was noted, and highly conserved residues in N-terminal region help in the formation of the two highly conserved domains such as PH domain and PTB domain. Results generated from this analysis will be more beneficial to researchers in understanding more about insulin signalling mechanism(s) as well as insulin resistance pathway. We discuss here that bioinformatics tools utilized in this study can play a vital role in addressing the complexity of structural topology to understand structure–function relationships in insulin signalling cascades.