Insulin signaling pathways
Cellular responses to insulin include the regulation of blood sugar levels by increased glucose uptake in muscle and fat; increased storage of energy reserves in fat, liver and muscle through the stimulation of lipogenesis, glycogen synthesis and protein synthesis; decreased glucose production by the liver and inhibition of the mobilization of stored energy reserves via lipolysis, glycogenolysis or protein breakdown. Insulin also acts as a growth factor and stimulates cell growth, differentiation and survival. Aberrant signaling by the insulin receptor exon 11 minus isoform (IR-A), which binds IGF-II with high affinity, is associated with some cancers. The 1992 Nobel Prize in Physiology or Medicine was awarded to Edwin Krebs and Edmond Fischer for their discovery that reversible phosphorylation of proteins is the key regulatory process in the transmission of signals that impinge on cells. That signaling pathways involve cascades of phosphorylation (by kinases) and dephosphorylation (by phosphatases) has been shown to be true not only for signaling by insulin and other growth factors, but for other types of stimuli as well.
Insulin receptor signaling starts with the autophosphorylation of key tyrosine residues in the intracellular region of the IR, generating phosphotyrosine docking sites for various proteins containing SH2 (Src-homology-2) domains or PTB (phosphotyrosine binding) domains. These docked substrates include enzymes and adaptors such as IRS proteins and Shc. Insulin signaling is downregulated by internalization of the insulin/IR complex leading to dissociation and degradation of insulin in the intracellular endosome/lysosome system, inactivation of the autophosphorylated IR by the phosphatase PTP1B and recycling of the inactivated IR back to the plasma membrane.
The players involved in orchestrating intracellular signaling pathways
As Brognard and Hunter point out, the regulation of intracellular signaling pathways is complex given there are 539 kinases in the human genome that act on a plethora of phosphorylation sites in hundreds of target proteins. All protein kinase catalytic domains have a common architecture and sequence alignments of kinase domains can be found at ref . Many of the proteins involved in insulin signaling are also involved in signaling from unrelated growth factors indicating that specific responses result from both qualitative and quantitative differences in the proteins involved at any one time. The situation is analogous to large orchestras comprised of a common set of instruments (the pool of intracellular signaling molecules) generating different outputs (the performance) depending on the conductor (growth factor) and the score at hand (growth factor receptor). One example is the involvement of PI3 kinase and the Ser/Thr protein kinase Akt in insulin-dependent glucose transport on the one hand and the majority of cancers on the other.
To help the reader with the following sections a list of the acronyms and abbreviations of some of the proteins involved in insulin signaling is provided at Acronyms for insulin signaling proteins.
Blood glucose levels are under tight control
As discussed in the section Glucose metabolism in normal individuals plasma glucose remains in a narrow range between 4 and 7 mM reflecting the balance between (i) the release of glucose into the circulation by either absorption from the intestine or the breakdown of stored glycogen in the liver and (ii) the uptake and metabolism of blood glucose by peripheral tissues.
Glucose transport into cells is via specialized proteins called glucose transporters (GLUTs), its rate of entry being limited by the number of glucose transporters on the cell surface and the affinity of the transporters for glucose. The basal glucose transporters (GLUT1 and GLUT3) are high affinity transporters and are present in nearly all cells. They have a Km for glucose of around 2-5 mM. Since this is less than the average blood glucose concentration of 5-7 mM, most tissues take up glucose at a fairly constant rate, regardless of the amount present in the blood.
Muscle and fat cells express a third type of glucose transporter, the high-affinity, insulin-responsive GLUT4, with a Km around 5 mM. As discussed below, insulin through its action on the insulin receptor is the primary regulator of blood glucose levels. Insulin decreases blood sugar levels by: (i) increasing glucose uptake in muscle and fat through triggering the translocation of the intracellular glucose transporter GLUT4 to the plasma membrane; (ii) stimulating the storage of glycogen and fat in muscle, liver and adipose tissues through stimulation of the synthesis of glycogen (Glycogenesis), fat (lipogenesis) and protein and (iii) reducing glucose production and release by the liver through inhibition of glycogen breakdown (Glycogenolysis). Insulin signaling also inhibits the breakdown of fat (lipolysis) and protein.
Autophosphorylation of the IR
There are 13 potential tyrosine phosphorylation sites in the intracellular insulin receptor β-chain that provide potential phosphotyrosine docking sites for SH2-containing and PTB-containing signaling proteins. These sites are distributed across the juxtamembrane, catalytic domain and C-tail regions of each IR monomer. Autophosphorylation is in trans where each kinase in the dimer phosphorylates the intracellular regions of its partner. Initial phosphorylation includes the highly conserved residue Tyr984 (IR-B numbering) in the juxtamembrane region and the three tyrosines in the activation loop (Tyr1158, Tyr1162, Tyr1163, IR-B numbering). Tyr984 interacts with several conserved residues in the N-terminal lobe of the IR kinase domain, stabilizing a non-productive position of the αC helix. This inhibitory interaction is released when Tyr984 is phosphorylated. Following phosphorylation of Tyr1158, Tyr1162 and Tyr1163 the activation loop is significantly displaced allowing unrestricted access to the kinase binding site for ATP and protein substrates including multiple phosphorylation sites on the intracellular region of its dimer partner.
Initial events, the binding and Tyr phosphorylation of IRS proteins or Shc
The insulin receptor substrate proteins IRS1 and IRS2 are key targets of the insulin receptor tyrosine kinase and are required for the hormonal control of metabolism. Phosphorylation of Tyr972 in the juxtamembrane region (IR-B numbering) provides a docking site for the IRS family of adaptors (IRS1, IRS2, IRS3 and IRS4) as well as the adaptor Shc. They are highly homologous yet appear to have overlapping and complementary functions influenced by the cell type in which they are expressed. IRS1 and IRS2 are large proteins (>1200 amino acids residues) with a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain at their N-terminus with the remainder of the protein (approx 1000 residues) largely unstructured. IRS1 contains 34 Tyr residues which if phosphorylated represent potential binding sites for various SH2-containing proteins including PI3K, Grb2 and Shc. The involvement of IRS1 and IRS2 in intracellular signaling is subjected to regulatory feedback through phosphorylation on multiple serine/threonine residues by a variety of Ser/Thr protein kinases. Approximately 70 of the 230 Ser/Thr residues in IRS1 lie in canonical kinase phosphorylation motifs. Both IRS1 and IRS2 but not Shc are involved in the PI3K/Akt signaling pathway affecting glucose uptake and metabolism. In contrast Shc binding directly to IR or indirectly via IRS-1 or IRS-2 initiates the Ras/MAP kinase pathway involved in gene expression and cell growth.
Metabolic signaling – the PI3K/Akt pathway
Activation of Akt
The canonical signaling pathways for IR are shown in the figure below. Only a generalized summary will be provided here. More detailed accounts can be found in the reviews. Phosphorylation of IRS-1 or IRS-2 provides docking sites for the SH2 domains of the regulatory subunit (p85) of PI3K which is bound to the catalytic subunit p110, as a heterodimer. The lipid products of PI3K are phosphatidylinositol  bisphosphate (PIP2) and phosphatidylinositol  triphosphate (PIP3) which induce the activation of protein serine kinase cascades by co-recruitment to membranes of phosphoinositide-dependent kinase-1 (PDK1) and its substrate kinases Akt/PKB and atypical protein kinase Cs (aPKCs), via their respective PH domains. PDK1 is believed to be constitutively active and does not require membrane lipid binding for the efficient phosphorylation of those substrates in the cytosol (not at the cell membrane). Binding to PIP2 and PIP3 is required for the activation of some substrates including Akt, which require a proper orientation of the kinase and PH domains of PDK1 and Akt at the membrane. PDK1 partially activates the serine kinases Akt/PKB and aPKC by phosphorylation of a conserved threonine residue (Thr308 in Akt) in the kinase regulatory loop. Full activation of Akt/PKB occurs following phosphorylation of a C-terminal hydrophobic motif containing Ser473, catalysed by a distinct enzyme, most probably mTORC2. The duration and amplitude of Akt signalling are controlled by the phosphatase PHLPP, that acts specifically on the hydrophobic phosphorylation motif.
Figure 1. Canonical insulin signaling pathways showing the principal components of the PI3K/Akt (metabolic) and Ras/MAP kinase (Mitogenic) pathways. Adapted from Siddle, 2011.[Click to enlarge]
Activated Akt/PKB phosphorylates multiple substrates and controls a variety of downstream responses depending on cell type. Well-established Akt/PKB substrates include GSK-3, regulating glycogen synthesis; FOXO transcription factors, which on phosphorylation move from the nucleus to the cytoplasm thus inhibiting foxo- dependent transcription of gluconeogenic and other genes; the Rheb GTPase activating complex TSC1/2 that regulates mTOR and protein synthesis; and the Rab GTPase activating protein AS160/TBC1D4, regulating glucose transport . The details of glucose transport regulation are presented in the article ‘Insulin-induced glucose transport ’.
Insulin regulation of glucose metabolism The liver, through its possession of GLUT2 as the major glucose transporter, is sensitive to changes in blood glucose levels as its Km (the concentration where glucose uptake is 50% of its maximum rate) is of the order 15-20 mM, well above the normal fasting level of 5-7 mM. In the fasting state blood glucose levels are maintained by the liver through the release of glucose generated by either Gluconeogenesis or Glycogenolysis. In humans gluconeogenesis is the more important process. In a 68 hour fast gluconeogenesis accounted for around 64% of total glucose production during the first 22 hours of fasting, 82% during the next 14 hours and 96% during the next 18 hours.
Insulin is the only hormone that lowers the concentration of blood glucose by regulating hepatic glucose metabolism. It stimulates glucose utilisation via Glycolysis and Glycogenesis and inhibits glucose production via gluconeogenesis and glycogenolysis. Through the Akt-signaling pathway, insulin increases glycolysis by upregulating the level of transcription of the enzymes that carry out the three rate-limiting steps in the pathway: the phosphorylation of glucose by hexokinase/glucokinase (step 1), the phosphorylation of fructose-6-phosphate by phosphofructokinase (step 3) and the transfer of phosphate from phosphoenolpyruvate to ADP by pyruvate kinase (step 10).
Insulin via Akt increases glycogenesis by activating glycogen synthase, the enzyme which adds glucose units to the growing polysaccharide chain of glycogen. Activated Akt phosphorylates and inactivates glucose synthase kinase 3 (GSK-3) and/or protein kinase A (PKA) preventing them from phosphorylating and inactivating glycogen synthase. Insulin also increases the levels of activated glycogen synthase by activating protein phosphatase 1 (PP1). PP1 is a Ser/Thr protein phosphatase which can dephosphorylate and activate glycogen synthase. Insulin does not activate PP1 globally but rather specifically targets discrete pools of PP1 localized at glycogen particles via a specific one of its regulatory subunits. The pathway by which insulin activates glycogen-associated PP1 remains to be established.
Insulin is known to modulate the expression of over 100 genes at the transcriptional level in mammals. The transcriptional effects of insulin are widespread and concern multiple biological phenomena. In the liver, the PI3 kinase/Akt pathway of insulin signaling activates the transcription factor sterol regulatory element binding protein-1c (STREBP-1c) which in turn increases the transcription of most of the genes encoding metabolic enzymes including the rate-limiting glycolytic enzymes glucokinase and pyruvate kinase and lipogenic enzymes such as fatty acid synthase and acetylCoA carboxylase. The genes that are inhibited by insulin are limited, and encode mainly enzymes involved in hepatic glucose production. Here through phosphorylation and translocation of the transcription factor FOXO out of the nucleus, insulin suppresses gluconeogensis by reducing the levels of expression of the three rate-limiting enzymes PEP carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. Gluconeogensis is the process by which glucose (a six carbon sugar) is synthesized from smaller 3-carbon components such as pyruvate and lactate. The pyruvate is generated from the breakdown of proteins and amino acids as well as by the oxidation of lactate. Lactate is produced by muscle tissues and is transported to the liver by the bloodstream.
Insulin suppresses the production of glucose by glycogenolysis, the sequential removal of glucose monomers as glucose-1-phosphate from glycogen (a polyglucoside) by the phosphorylated form of the enzyme glycogen phosphorylase (phosphorylase a). The glucose-1-phosphate is then converted to glucose-6-phosphate by the enzyme phosphoglucomutase. In liver cells the main purpose of the breakdown of glycogen is for the release of glucose into the bloodstream for uptake by other cells. The phosphate group of glucose-6-phosphate is removed by G6Pase and the free glucose exiting the cell via GLUT2. In muscle cells G6Pase is absent and role of glycogen degradation is to provide an immediate source of glucose-6-phosphate for glycolysis, to provide energy for muscle contraction. Insulin inhibits glycogenolysis by activating PP1 and the enzyme phosphodiesterase. Activated PP1 directly dephosphorylates glycogen phosphorylase a, reforming the inactive glycogen phosphorylase b. The phosphodiesterase converts cAMP to AMP, thus inactivating PKA and blocking the phosphorylation cascade that ends with formation of active, phosphorylated glycogen phosphorylase a.
Growth signaling – the Ras/MAP kinase pathway
In common with many receptor tyrosine kinases, IR and IGFR regulate cell growth-related gene expression via the Ras/MAP kinase pathway. The pathway is initiated by recruitment of the adaptor/guanine nucleotide exchange factor complex Grb2/SOS to phosphorylated Shc and/or IRSs. It is unclear whether Shc-bound and IRS-bound Grb2/SOS complexes are equally effective activators of the small GTPase Ras, given the differences in their abundance, subcellular localisation and potential co-recruitment of additional components. In some cells, Shc is the more important substrate for Ras/MAP kinase activation, while in others, IRS-dependent pathways appear to predominate. Shc and IRSs may compete in binding to IR and in recruiting a limited pool of Grb2, and this could influence signaling to ‘metabolic’ versus ‘mitogenic’ responses.
When Ras is activated by incoming signals, it initiates a phosphorylation cascade activating the Ser/Thr kinase Raf (ERK1, a MAP kinase kinase kinase), which phosphorylates and activates the dual specificity Tyr/Thr kinase MEK (a MAP kinase kinase), which in turn phosphorylates and activates the Ser/Thr kinase MAP Kinase. MAP kinase then phosphorylates various substrates which increase the transcription of genes, and the synthesis of proteins involved in cell growth, differentiation and survival. Scaffold proteins play a role in co-ordinating this cascade, and may influence cellular responses through effects on signal intensity and duration, localisation of complexes and recruitment of modulatory proteins such as phosphatases and ubiquitin ligases.
Ras-GTPases are considered inactive when bound to guanosine diphosphate (GDP), and active when bound to guanosine triphosphate (GTP). When SOS binds Ras, it causes Ras to release its bound nucleotide (usually GDP) and the SOS-Ras complex falls apart. Once released from SOS, Ras generally binds fresh GTP from the cytosol (since it is 10 times more abundant than GDP) and is activated. Ras acts as a molecular switch that binds to downstream effectors, such as the protein kinase Raf (ERK1, MAP kinase kinase kinase), and localises them to the membrane resulting in their activation.
Raf is a multidomain Ser/Thr protein kinase whose additional domains aid the regulation of its catalytic activity. N-terminally there is a Ras-binding domain (RBD) and a C-kinase homologous domain 1 (C1 domain) adjacent to each other which by direct physical interaction act as a single autoinhibitory unit to negatively regulate the activity of the protein kinase domain. The C-terminal half of c-Raf folds into a single protein kinase domain, responsible for catalytic activity. Between the autoinhibitory unit and the kinase domain is a long unstructured, flexible region that is highly enriched in Ser residues. Its most likely role is to act as a natural "hinge" between the rigidly folded autoinhibitory unit and the catalytic domain, enabling complex movements and profound conformational rearrangements within the molecule.
The activation of Raf includes three key steps: Ras binding, dimerisation and activation loop transphosphorylation. The most important inhibition of Raf involves the direct, physical association of the N-terminal autoinhibitory unit (RBD plus C1 domain) to the kinase domain, which results in the occlusion of the catalytic site and no kinase activity. This "closed" state is relieved when the autoinhibitory domain of Raf engages activated GTP-bound Ras, resulting in a conformational change ("opening") of the Raf molecule enabling kinase activation and substrate binding. Kinase activation requires dimerisation of two ‘open’ Raf molecules resulting in the correct positioning of the activation loops (that control the catalytic activity of all known protein kinases) in an active-like conformation in the dimer. The Raf dimers are symmetric and have two, partially active catalytic sites. At this stage, the activity of Raf kinase is low, and unstable.
Full activity and stabilization of the active state occurs following phosphorylation of the activation loops by another Raf dimer. Due to the architecture of the dimers, this phosphorylation can only take place in trans (i.e. one dimer phosphorylates another, in a four-membered transitional complex). By interacting with conserved Arg and Lys residues in the kinase domain, the phosphorylated activation loops shift conformation and become ordered, permanently locking the kinase domain into a fully active state until dephosphorylated.
While activated, Raf can act on many substrates the most important target is MAP kinase kinase (also called MEK). Raf phosphorylates MAP kinase kinase on at least two sites in its activation loop. Activated MEK in turn activates MAP kinase (ERK 2) by phosphorylating a conserved Thr residue in its activation loop. MAPK (ERK2) has numerous substrates in cells and is also capable of translocating into the nucleus to activate nuclear transcription factors. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, cell migration, inhibition of apoptosis, and cell differentiation.
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