Information

C9. Insulin Signaling - PI3K and Akt (Protein Kinase B) - Biology

C9.  Insulin Signaling - PI3K and Akt (Protein Kinase B) - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Type 2 diabetes, in which people become resistant to insulin which leads to high blood sugar and the cascade of health consequences that follow, is pandemic in the world. The other, Insulin Receptor Substrate 1, IRS1, a "scaffolding protein" leads to the movement of the GLUT4 protein (Glucose transport protein) to the cell surface. These activities are shown schematically in the figure below.

Figure: Initial Signaling on Binding of Insulin to the Insulin Receptor

After phosphorylation by the activated insulin receptor protein tyrosine kinase, IRS-1 binds phosphatidylinositol 3-kinase (PI3K) that causes phosphorylation of the 3'OH on phosphatidyl inositol (PI) in the inner leaflet of the membrane to form PI(3)P. PI3K is a member of a family of kinases that phosphorylates pI. The metabolic pathway centered on pI3K is one of the most mutated in human cancers. PI(3)P in turn recruits to the membrane other inactive kinases, phosphoinositide-dependent kinase 1, PDK1 and Akt, also known as PKB.

Figure: Phosphorylated Phosphatidylinositol derivatives

On binding of PI(3)P, PDK1 becomes an active kinase, which phosphorylates and activates Akt. The family of three Akt kinases are major Ser/Thr protein kinase that phosphorylates proteins involved in a host of cell activities, including regulation of glucose transport, cell proliferation and death. In the insulin signaling pathway, active (phosphorylated) Akt leads to movement of the GLUT4 protein from intracellular endosomal vesicles to the cell surface, which offers a quicker way to import glucose into the cell that if Akt activated GLUT 4 gene expression.

pi3K Pathway: Animation from Promega

Contributors

  • Prof. Henry Jakubowski (College of St. Benedict/St. John's University)

The Insulin Signaling Pathway

Insulin is a peptide hormone that predominantly functions to reduce blood glucose levels. It is secreted from beta cells found in the islets of the pancreas in response to nutrient uptake and increased blood glucose levels. When insulin binds to its receptors on target cells, such as skeletal muscle cells and adipocytes, a signaling cascade is initiated, which culminates in the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell membrane. Once GLUT4 is incorporated into the plasma membrane, it functions to promote the uptake of extracellular glucose, which is then stored as glycogen in these cells, thereby regulating blood glucose [1] .

Insulin also regulates blood sugar through inhibiting gluconeogenesis (de novo glucose production) and glycogenolysis (glycogen breakdown) in the liver. Besides regulating blood glucose levels, insulin also plays critical roles in facilitating protein and lipid synthesis and preventing the conversion of protein and fat to glucose.

While insulin is widely viewed as a glucose homeostasis regulating hormone, an increasing body of research is illuminating broader roles for this peptide. Insulin signaling pathways are highly conserved, with insulin-like signaling systems found in all metazoans, and they have been shown to regulate many evolutionarily conserved processes, including lifespan and reproduction [2] .

The Insulin Receptor

The insulin receptor belongs to the superfamily receptor tyrosine kinases (RTKs) [3,4] and is activated by insulin, as well as insulin-like growth factors (IGF1-2). It is a heterotetrametric protein consisting of two extracellular α subunits and two transmembrane β subunits, which are linked together by disulfide bonds. Most RTKs bind directly to signaling proteins. The insulin receptor, however, binds to phosphorylated residues on partner proteins, namely a family of large docking proteins known as the insulin receptor substrate family (IRS1-6) [5,6] , as well as the adapter protein Shc (Src homology 2 domain containing) [7] .

Insulin Receptor Pathways

When insulin binds to the extracellular α subunits of the insulin receptor, a conformational change is induced, which then results in the autophosphorylation of several tyrosine residues present in the β subunits. These form the binding sites for IRS proteins, which contain phosphotyrosine (PTB) binding domains, or for Shc adapter proteins, containing src-homology 2 (SH2) domains. Binding of the insulin receptor to either IRS or Shc forms a platform that allows for the assembly of a signal transduction particle that gives rise to multiple intracellular signaling pathways [8] .

Two principle pathways result from the insulin receptor-IRS interaction, the PI3K/AKT (also known as protein kinase B or PKB) pathway, and the Ras/MAPK (also known as extracellular signal regulated kinase or ERK). The PI3K (phosphoinositol 3-kinase) pathway is linked exclusively through IRS and is responsible for most of insulin's metabolic effects in the cell [9,10] . The MAPK pathway, on the other hand, stems from IRS, as well as Shc, and is involved in the regulation of gene expression and, in cooperation with the PI3K pathway, also regulates cell growth and differentiation [11] .

PI3K / AKT Pathway

The PI3K pathway is activated by the binding of PI3K regulatory subunits p85 and p55 to IRS1 and IRS2. This results in the activation of the PI3K catalytic subunit, p110. Once the p110 subunit is activated, PI3K then catalyzes the phosphorylation of phosphatidylinositol (PI) to generate PIP3 (phosphatidylinositol 3,4,5-triphosphate) at the cell membrane [10,12] . PIP3 is an important second messenger that functions to recruit PDK1 (3-phosphoinositide dependent protein kinase-1) and AKT to the membrane, where phosphorylation of PDK1 then activates the serine/threonine residues of AKT [13] . From here, AKT plays a role in four critical downstream processes.

AKT is involved in the regulation of protein synthesis via the substrate protein mTOR, a serine/threonine kinase that functions as a nutrient sensor. mTOR stimulates protein synthesis through the phosphorylation of 4EBP1 (eukaryotic translation initiation factor 4E-binding protein 1) and p70S6K (p70 ribosomal protein S6-kinase) [14] .

AKT acts in the regulation of glycogen synthesis via glycogen synthase kinase 3 (GSK3), another serine/threonine kinase, which, amongst other roles, functions to inhibit glycogen synthase. GSK3 is inhibited when phosphorylated by AKT/PKB, which results in glycogen synthesis [15] .

AKT plays a role in the regulation of gluconeogenic and adipogenic genes through the transcription factor FOXO1 (forkhead box-containing protein 1, subfamily O). In the absence of insulin, FOXO1 translocates to the nucleus where it activates the expression of genes involved in gluconeogenesis, such as phosphoenolpyruvate carboxykinase (PEPCK) [13] . It also activates the expression of cyclin G2, an atypical cyclin that blocks the cell cycle, which is inhibited by insulin [16] , and appears to play a key role in insulin-induced mitogenesis. When phosphorylated by AKT, FOXO1 is sequestered in the cytoplasm, and therefore cannot activate the expression of its target genes.

Importantly, AKT also regulates translocation of the insulin-sensitive glucose transporter GLUT4, which is sequestered in intracellular vesicles of muscle cells and adipocytes to the cell membrane via exocytosis, where it facilitates the uptake of glucose from the blood into cells. This is achieved through the phosphorylation of AS160 (160-kDa AKT substrate), a GTPase-activating protein that activates RAB, a small G protein involved in membrane trafficking by blocking the exchange of GTP for GDP [17] .

Figure 1: The PI3K and MAPK pathways.

Figure 2: The translocation of GLUT4 in the PI3K and MAPK pathways.

Ras / MAPK Pathway

The MAPK pathway is an essential secondary branch of the insulin signaling pathway. It is activated independently of the PI3K pathway either through binding of growth factor receptor-bound protein 2 (Grb2) to tyrosine-phosphorylated Shc, or through Sh2 binding to the insulin receptor. The amino-terminal SH3 domain of Grb2 binds to proline-rich regions of proteins such as son-of-sevenless (SOS), a guanine nucleotide exchange factor that catalyzes the shift of membrane-bound Ras from an inactive form (Ras-GDP) to an active form (Ras-GTP) [18] . Activated Ras-GTP is then able to stimulate downstream effectors, such as the Serine/Threonine kinase Raf, which activates its downstream targets MEK1 and MEK2 that go on to phosphorylate and activate the MAP kinases Extracellular signal-regulated kinase 1/2 (ERK1/2). Activated ERK1/2 are directly involved in multiple cellular processes, ranging from cell proliferation and differentiation. They act by regulating gene expression as well as extra-nuclear events, such as cytoskeletal reorganization, through the phosphorylation and activation of target proteins in both the cytosol and nucleus [11] .

Negative Regulation of Insulin Receptor Signaling and Signal Termination

Many mechanisms exist to attenuate, finetune, and terminate insulin signaling, both at the level of the receptor and at various points in the cascade. The insulin receptor and IRS proteins are negatively regulated by multiple systems, such as ligand-induced downregulation, tyrosine protein phosphatases, and serine phosphorylation. Phosphatases also regulate the subsequent steps in the associated protein kinase cascades.

Negative Feedback Loops in Response to Insulin

Negative feedback loops have been shown to play an essential role in finetuning this complex network [13,2] . Chronic exposure to insulin (hyperinsulinemia) results in a decrease of insulin receptors on the cell surface [19] , as well as decreased IRS1 and IRS2 in vitro and in vivo in mice, which has been linked to insulin resistance in animal models [13] . The decrease in insulin receptors occurs through endocytosis by clathrin-coated vesicles. These receptors are then recycled or degraded within the lysosomes of the cell [20] . Receptor endocytosis has since been demonstrated to be a critical negative feedback mechanism that is relevant to the entire class of RTKs.

IRS signaling is negatively regulated by serine phosphorylation and kinases, such as ERK, S6 kinase, and c-Jun-N-terminal kinase (JNK), which are all activated by insulin. This is another negative feedback mechanism in the insulin signaling pathway [13] . The receptor for TNFα (TNFR), which predominantly functions in apoptosis and inflammation, induces IRS1 serine phosphorylation through JNK [13] , causing insulin resistance in vitro, and in vivo in animal models as well as humans [21] .

Attenuation of Insulin Signaling by Protein and Phospholipid Phosphatases

PTP1B is a major protein tyrosine phosphatase that dephosphorylates the insulin receptor. This protein resides in the endoplasmic reticulum and acts on the insulin receptor during internationalization and recycling of the receptor to the plasma membrane [22,23] . PTP1B also acts to dephosphorylate residues on activated IGF-1R and IRS proteins to reduce their activity. PTP1B knockout mice have been shown to be more sensitive to insulin and exhibit improved glucose tolerance [24,25] .

The serine/threonine protein phosphatase 1 (PP1) is known to play a role in both glucose and lipid metabolism through the regulation of multiple rate-limiting enzymes, including glycogen synthase, hormone-sensitive lipase, or acetyl CoA carboxylase [26] . Protein phosphatase 2A (PP2A) also plays a critical role in regulating the activities of many protein kinases involved in the insulin cascade, including Akt, PKC, and ERK [27] . Interestingly, PP2A has been demonstrated to be hyperactivated in diabetic states [28] .

Protein phosphatases 2B (PP2B), another serine/threonine phosphatase also known as calcineurin, has been shown to dephosphorylate Akt [29] . PH domain leucine-rich repeat protein phosphatases PHLPP-1 and PHLPP-2, members of the PP2C family, act to dephosphorylate both Akt and PKCs [30] . When PHLPP1 is over expressed in cells, the function of Akt and GSK3 activity is reduced. This results in a decrease in glycogen synthesis and glucose transport [31] . Obese and diabetic patients have been shown to have elevated levels of PHLPP1 in both adipose tissue and skeletal muscle which correlates with decreased Akt2 phosphorylation [31,32] .

Negative regulation of the PI3K pathway occurs through dephosphorylation and subsequent inactivation of PIP3 by phospholipid phosphatases such as the tumor suppressor PTEN (phosphatase and tensin homolog) and SHIP2 (SH2-containing inositol 5'-phosphatase-2). PTEN dephosphorylates phosphoinositides on the 3'-position, whereas SHIP2 functions at the 5'-position [33] .

Other Negative Modulators of Insulin Receptor Signaling

Suppressor of Cytokine Signaling (SOCS) proteins also function to attenuate insulin receptor signaling. These are mediators of cytokine receptor signaling, such as leptin and IL-6 receptors that act through Janus kinases (JAK) and signal transduction, as well as activation of transcription (STAT) proteins [34,35] . SOCS1, SOCS3, SOCS6, and SOCS7 act by binding to the insulin receptor to inhibit signaling, as well as by targeting IRS-1 and IRS-2 for proteasomal degradation [35] .

Figure 3: Negative regulators of the insulin signaling pathway.

Dysregulated Insulin Signaling and Disease

Type 2 diabetes is the primary disease associated with insulin and the insulin signaling pathways. This complex and heterogeneous disorder is caused by a combination of lifestyle and environmental factors, such as the typical western diet (which is high in fats and sugars), inactivity, and obesity, and is further modified by various genetic determinants [36] . Type 2 Diabetes is caused by two factors, insulin sensitivity (or insulin resistance) attributed to dysregulation of the insulin receptor signaling cascade, and changes in the production and secretion of insulin by the beta cells of the pancreas in response to elevated glucose. However, the relative impact of both defects on the development of diabetes has not yet been ascertained, nor have the specific molecular events at the tissue and cellular level [2] . As insulin receptors are present on many different cell types, dysregulation of the insulin signaling network effects multiple organs of the body in diabetes.

Thrombosis and Atherosclerosis

Heart attacks and strokes, precipitated by pathological blood clots (thrombi), are the leading cause of death in diabetic patients. The reason for this is twofold firstly, patients with diabetes have an increased risk of developing more extensive atherosclerosis (AS) [37] , and secondly, they possess "hyperactive" platelets, which are prone to forming thrombi. The rupture of an atherosclerotic plaque, combined with this augmented propensity for platelets to form large occlusive thrombi, increases the risk of fatal thrombotic events in diabetic individuals. Endothelial dysfunction, as well as the hyperactive phenotype of diabetic platelets, are well reported [38,39,40] , but the exact underlying mechanisms remain largely unknown.

Diabetic patients also have an increased risk of developing Alzheimer's Disease (AD), a neurodegenerative disorder, although the exact relationship between these two diseases is poorly understood. Insulin signaling dysfunction has been reported in the AD brain, however, whether this is a cause or consequence of the disease has not yet been ascertained [41,42] .

There is growing evidence that abnormal insulin levels and dysregulated insulin signaling lead to cancer development and progression. A higher incidence of cancer is found in obese patients and those with type 2 diabetes. Many of the proteins that play a role in the insulin signaling pathways are involved in promoting cell proliferation and mitosis, as well as preventing apoptosis, which may increase the risk of tumor formation and metastasis [43] .

Despite the tremendous progress made in understanding insulin and insulin receptor signaling over the last decades, there is still much left to be uncovered regarding how these complex networks regulate cells in both normal and disease states.

Recommended Products

We offer a wide range of research tools that be used for studing the insulin signalling pathway, glucose storage, glucose uptake, and protein lipid synthesis through Ras, Akt, mTor and MAPK. Below we have listed some of our most popular antibodies and immunoassays.


Department of Medicine, Division of Endocrinology, Metabolism and Nutrition, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

Albert Einstein-Mount Sinai Diabetes Research Center and the Fleischer Institute for Diabetes and Metabolism, Albert Einstein College of Medicine, Bronx, NY, USA

Department of Medicine and Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA

Department of Medicine, Division of Endocrinology, Metabolism and Nutrition, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

Albert Einstein-Mount Sinai Diabetes Research Center and the Fleischer Institute for Diabetes and Metabolism, Albert Einstein College of Medicine, Bronx, NY, USA

Department of Medicine and Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA

Emeritus Senior Scientist Emeritus Professor of Physiology Emeritus Professor of Medicine

National Institutes of Health, Bethesda, MD, USA

Tufts University School of Medicine, Boston, MA, USA

Albert Einstein College of Medicine, Bronx, NY, USA

Distinguished NIH Scientist Emeritus

Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD, USA

Ensign Professor of Medicine

Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA

Vincent Astor Distinguished Professor of Medicine, Chief Co-Director

Division of Gastroenterology and Hepatology, Joan & Sanford I. Weill Department of Medicine, Weill Cornell Medical College

Center for Advanced Digestive Care, New York-Presbyterian Hospital and Weill Cornell Medical Center, New York, NY, USA

Professor of Medicine Associate Director

Cell Biology & Pathology, Albert Einstein College of Medicine

Marion Bessin Liver Research Center, Bronx, NY, USA

Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bronx, NY, USA

The Herman Lopata Chair in Liver Disease Research Professor of Medicine and Anatomy and Structural Biology Associate Chair of Medicine for Research Chief, Division of Hepatology Director, Marion Bessin Liver Research Center

Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY, USA

Summary

Insulin signaling is initiated by the activation of the insulin receptor (IR) through autophosphorylation of the tyrosine residue in the IR, and then many signaling molecules including IR substrate 1 and 2, phosphoinositide-3-kinase (PI3K) and AKT/protein kinase B (PKB) are involved to modulate downstream signaling pathways. This chapter focuses on the role of PI3K and AKT/PKB in insulin signaling related to pathophysiology in type 2 diabetes mellitus, non-alcoholic fatty liver disease, and liver cancer. To understand the physiologic role of the signaling molecules in insulin action and glucose homeostasis, the chapter reviews the biochemical characteristics of the PI3K and AKT kinases. The secretion of insulin and glucagon are tightly regulated to modulate gluconeogenesis and lipogenesis in hepatocytes, and dysregulation of the proximal signaling pathways of the ligands results in the hyperglycemia and hyperlipidemia seen in metabolic diseases.


Role of PI3K/AKT, cPLA2 and ERK1/2 signaling pathways in insulin regulation of vascular smooth muscle cells proliferation

Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.


References

Avruch, J. Insulin signal transduction through protein kinase cascades. Mol. Cell Biochem. 182, 31–48 (1998).

Ullrich, A. & Schlessinger, J. Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212 (1990).

Elchebly, M. et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283, 1544–1548 (1999).

Ueki, K., Kondo, T. & Kahn, C. R. Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms. Mol. Cell Biol. 24, 5434–5446 (2004).

Emanuelli, B. et al. SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-α in the adipose tissue of obese mice. J. Biol. Chem. 276, 47944–47949 (2001).

Friedman, J. E. et al. Reduced insulin receptor signaling in the obese spontaneously hypertensive Koletsky rat. Am. J. Physiol. 273, E1014–E1023 (1997).

Sun, X. J. et al. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature 352, 73–77 (1991).

Sun, X. J. et al. Role of IRS-2 in insulin and cytokine signalling. Nature 377, 173–177 (1995).

Lavan, B. E., Lane, W. S. & Lienhard, G. E. The 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor substrate family. J. Biol. Chem. 272, 11439–11443 (1997).

Fantin, V. R. et al. Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells. J. Biol. Chem. 273, 10726–10732 (1998).

Cai, D., Dhe-Paganon, S., Melendez, P. A., Lee, J. & Shoelson, S. E. Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. J. Biol. Chem. 278, 25323–25330 (2003).

Lehr, S. et al. Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro. Biochemistry 39, 10898–10907 (2000).

Baumann, C. A. et al. CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 407, 202–207 (2000).

Gustafson, T. A., He, W., Craparo, A., Schaub, C. D. & O'Neill, T. J. Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain. Mol. Cell Biol. 15, 2500–2508 (1995).

Virkamaki, A., Ueki, K. & Kahn, C. R. Protein–protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J. Clin. Invest. 103, 931–943 (1999).

Myers, M. G. Jr et al. The COOH-terminal tyrosine phosphorylation sites on IRS-1 bind SHP-2 and negatively regulate insulin signaling. J. Biol. Chem. 273, 26908–26914 (1998).

Algenstaedt, P., Antonetti, D. A., Yaffe, M. B. & Kahn, C. R. Insulin receptor substrate proteins create a link between the tyrosine phosphorylation cascade and the Ca2 + -ATPases in muscle and heart. J. Biol. Chem. 272, 23696–23702 (1997).

Fei, Z. L., D'Ambrosio, C., Li, S., Surmacz, E. & Baserga, R. Association of insulin receptor substrate 1 with simian virus 40 large T antigen. Mol. Cell Biol. 15, 4232–4239 (1995).

Zick, Y. Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance. Sci. STKE 2005, PE4 (2005).

Bouzakri, K. et al. Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes 52, 1319–1325 (2003).

Harrington, L. S. et al. The TSC1–2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J. Cell Biol. 166, 213–223 (2004).

Miller, B. S. et al. Activation of cJun NH2-terminal kinase/stress-activated protein kinase by insulin. Biochemistry 35, 8769–8775 (1996).

Cai, D. et al. Local and systemic insulin resistance resulting from hepatic activation of IKK-β and NF-κB. Nature Med. 11, 183–190 (2005).

Hirosumi, J. et al. A central role for JNK in obesity and insulin resistance. Nature 420, 333–336 (2002).

Aguirre, V., Uchida, T., Yenush, L., Davis, R. & White, M. F. The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J. Biol. Chem. 275, 9047–9054 (2000). An important paper that established the connection between Ser307 phosphorylation, JNK and insulin resistance.

Craparo, A., Freund, R. & Gustafson, T. A. 14-3-3 (ε) interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner. J. Biol. Chem. 272, 11663–11669 (1997).

Bard-Chapeau, E. A. et al. Deletion of Gab1 in the liver leads to enhanced glucose tolerance and improved hepatic insulin action. Nature Med. 11, 567–571 (2005).

Hirashima, Y. et al. Insulin down-regulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway. J. Endocrinol. 179, 253–266 (2003).

Rui, L., Yuan, M., Frantz, D., Shoelson, S. & White, M. F. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J. Biol. Chem. 277, 42394–42398 (2002).

Shimomura, I. et al. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol. Cell 6, 77–86 (2000).

Sesti, G. et al. Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J. 15, 2099–2111 (2001).

Araki, E. et al. Alternative pathway of insulin signaling in targeted disruption of the IRS-1 gene. Nature 372, 186–190 (1994).

Kubota, N. et al. Insulin receptor substrate 2 plays a crucial role in β cells and the hypothalamus. J. Clin. Invest. 114, 917–927 (2004).

Withers, D. J. et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391, 900–904 (1998).

Tseng, Y. H. et al. Prediction of preadipocyte differentiation by gene expression reveals role of insulin receptor substrates and necdin. Nature Cell Biol. 7, 601–611 (2005).

Miki, H. et al. Essential role of insulin receptor substrate 1 (IRS-1) and IRS-2 in adipocyte differentiation. Mol. Cell Biol. 21, 2521–2532 (2001).

Huang, C., Thirone, A. C., Huang, X. & Klip, A. Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes. J. Biol. Chem. 280, 19426–19435 (2005).

Taniguchi, C. M., Ueki, K. & Kahn, R. Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism. J. Clin. Invest. 115, 718–727 (2005).

Sun, X. J. et al. The IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter for insulin and cytokine action. Mol. Endocrinol. 11, 251–262 (1997).

Miura, A. et al. Insulin substrates 1 and 2 are corequired for activation of atypical protein kinase C and Cbl-dependent phosphatidylinositol 3-kinase during insulin action in immortalized brown adipocytes. Biochemistry 43, 15503–15509 (2004).

Tsuruzoe, K., Emkey, R., Kriauciunas, K. M., Ueki, K. & Kahn, C. R. Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling. Mol. Cell Biol. 21, 26–38 (2001).

Inoue, G., Cheatham, B., Emkey, R. & Kahn, C. R. Dynamics of insulin signaling in 3T3-L1 adipocytes. Differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2. J. Biol. Chem. 273, 11548–11555 (1998).

Ogihara, T. et al. Insulin receptor substrate (IRS)-2 is dephosphorylated more rapidly than IRS-1 via its association with phosphatidylinositol 3-kinase in skeletal muscle cells. J. Biol. Chem. 272, 12868–12873 (1997).

Sawka-Verhelle, D., Tartare-Deckert, S., White, M. F. & Van Obberghen, E. Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591–786. J. Biol. Chem. 271, 5980–5983 (1996).

Myers, M. G. Jr et al. IRS-1 activates phosphatidylinositol 3′-kinase by associating with src homology 2 domains of p85. Proc. Natl Acad. Sci. USA 89, 10350–10354 (1992).

Cheatham, B. et al. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell Biol. 14, 4902–4911 (1994).

Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα. Curr. Biol. 7, 261–269 (1997). Describes the discovery of PDK1.

Le Good, J. A. et al. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281, 2042–2045 (1998).

Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 (2005).

Maehama, T. & Dixon, J. E. PTEN: a tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol. 9, 125–128 (1999).

Wijesekara, N. et al. Muscle-specific Pten deletion protects against insulin resistance and diabetes. Mol. Cell Biol. 25, 1135–1145 (2005).

Tang, X., Powelka, A. M., Soriano, N. A., Czech, M. P. & Guilherme, A. PTEN, but not SHIP2, suppresses insulin signaling through the phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 adipocytes. J. Biol. Chem. 280, 22523–22529 (2005).

Sleeman, M. W. et al. Absence of the lipid phosphatase SHIP2 confers resistance to dietary obesity. Nature Med. 11, 199–205 (2005).

Shepherd, P. R., Withers, D. J. & Siddle, K. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem. J. 333, 471–490 (1998).

Yu, J. et al. Regulation of the p85/p110 phosphatidylinositol 3′-kinase: stabilization and inhibition of the p110α catalytic subunit by the p85 regulatory subunit. Mol. Cell Biol. 18, 1379–1387 (1998). Helped to define the molecular and functional relationships between the regulatory and catalytic subunits of PI3K.

Asano, T. et al. p110β is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity. J. Biol. Chem. 275, 17671–17676 (2000).

Tanti, J. F., Gremeaux, T., Van Obberghen, E. & Le Marchand-Brustel, Y. Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase. Biochem. J. 304, 17–21 (1994).

Tanti, J. F., Gremeaux, T., van Obberghen, E. & Le Marchand-Brustel, Y. Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. J. Biol. Chem. 269, 6051–6057 (1994).

Antonetti, D. A., Algenstaedt, P. & Kahn, C. R. Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain. Mol. Cell Biol. 16, 2195–2203 (1996).

Ueki, K. et al. Increased insulin sensitivity in mice lacking p85β subunit of phosphoinositide 3-kinase. Proc. Natl Acad. Sci. USA 99, 419–424 (2002).

Terauchi, Y. et al. Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 α subunit of phosphoinositide 3-kinase. Nature Genet. 21, 230–235 (1999). The first description of the paradoxical negative regulation of insulin signalling by the regulatory subunit p85α.

Chen, D. et al. p50α/p55α phosphoinositide 3-kinase knockout mice exhibit enhanced insulin sensitivity. Mol. Cell Biol. 24, 320–329 (2004).

Fruman, D. A. et al. Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 α. Nature Genet. 26, 379–382 (2000).

Mauvais-Jarvis, F. et al. Reduced expression of the murine p85α subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J. Clin. Invest. 109, 141–149 (2002).

Barbour, L. A. et al. Human placental growth hormone increases expression of the p85 regulatory unit of phosphatidylinositol 3-kinase and triggers severe insulin resistance in skeletal muscle. Endocrinology 145, 1144–1150 (2004). Shows that the physiologic upregulation of p85α in vivo induces insulin resistance.

Bandyopadhyay, G. K., Yu, J. G., Ofrecio, J. & Olefsky, J. M. Increased p85/55/50 expression and decreased phosphatidylinositol 3-kinase activity in insulin-resistant human skeletal muscle. Diabetes 54, 2351–2359 (2005).

Ueki, K., Algenstaedt, P., Mauvais-Jarvis, F. & Kahn, C. R. Positive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene products of the p85α regulatory subunit. Mol. Cell Biol. 20, 8035–8046 (2000).

Luo, J., Field, S. J., Lee, J. Y., Engelman, J. A. & Cantley, L. C. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex. J. Cell Biol. 170, 455–464 (2005).

Ueki, K. et al. Positive and negative roles of p85 α and p85 β regulatory subunits of phosphoinositide 3-kinase in insulin signaling. J. Biol. Chem. 278, 48453–48466 (2003). The first published connection between the p85α regulatory subunit and JNK activation.

Carpenter, C. L. & Cantley, L. C. Phosphoinositide kinases. Curr. Opin. Cell Biol. 8, 153–158 (1996).

Fang, D. & Liu, Y. C. Proteolysis-independent regulation of PI3K by Cbl-b-mediated ubiquitination in T cells. Nature Immunol. 2, 870–875 (2001).

Zheng, Y., Bagrodia, S. & Cerione, R. A. Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J. Biol. Chem. 269, 18727–18730 (1994).

Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M. & Hemmings, B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789 (1995).

Frame, S. & Cohen, P. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359, 1–16 (2001).

Sano, H. et al. Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J. Biol. Chem. 278, 14599–14602 (2003).

Harris, T. E. & Lawrence, J. C. Jr. TOR signaling. Sci. STKE 2003, RE15 (2003).

Tran, H., Brunet, A., Griffith, E. C. & Greenberg, M. E. The many forks in FOXO's road. Sci. STKE 2003, RE5 (2003).

Puigserver, P. et al. Insulin-regulated hepatic gluconeogenesis through FOXO1–PGC-1α interaction. Nature 423, 550–555 (2003).

Nakae, J. et al. The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Dev. Cell 4, 119–129 (2003).

Wolfrum, C., Asilmaz, E., Luca, E., Friedman, J. M. & Stoffel, M. Foxa2 regulates lipid metabolism and ketogenesis in the liver during fasting and in diabetes. Nature 432, 1027–1032 (2004).

Brazil, D. P., Yang, Z. Z. & Hemmings, B. A. Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem. Sci. 29, 233–242 (2004).

Gao, T., Furnari, F. & Newton, A. C. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol. Cell 18, 13–24 (2005).

Du, K., Herzig, S., Kulkarni, R. N. & Montminy, M. TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science 300, 1574–1577 (2003).

Koo, S. H. et al. PGC-1 promotes insulin resistance in liver through PPAR-α-dependent induction of TRB-3. Nature Med. 10, 530–534 (2004).

Chen, W. S. et al. Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev. 15, 2203–2208 (2001).

Cho, H., Thorvaldsen, J. L., Chu, Q., Feng, F. & Birnbaum, M. J. Akt1/PKBα is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J. Biol. Chem. 276, 38349–38352 (2001).

Cho, H. et al. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ). Science 292, 1728–1731 (2001).

George, S. et al. A family with severe insulin resistance and diabetes due to a mutation in AKT2. Science 304, 1325–1328 (2004).

Tschopp, O. et al. Essential role of protein kinase B γ (PKB γ/Akt3) in postnatal brain development but not in glucose homeostasis. Development 132, 2943–2954 (2005).

Chan, T. O., Rittenhouse, S. E. & Tsichlis, P. N. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu. Rev. Biochem. 68, 965–1014 (1999).

Bae, S. S., Cho, H., Mu, J. & Birnbaum, M. J. Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B. J. Biol. Chem. 278, 49530–49536 (2003).

Jiang, Z. Y. et al. Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc. Natl Acad. Sci. USA 100, 7569–7574 (2003).

Calera, M. R. et al. Insulin increases the association of Akt-2 with Glut4-containing vesicles. J. Biol. Chem. 273, 7201–7204 (1998).

Yamada, E. et al. Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles. J. Cell Biol. 168, 921–928 (2005).

Masure, S. et al. Molecular cloning, expression and characterization of the human serine/threonine kinase Akt-3. Eur. J. Biochem. 265, 353–360 (1999).

Standaert, M. L., Bandyopadhyay, G., Kanoh, Y., Sajan, M. P. & Farese, R. V. Insulin and PIP3 activate PKC-ζ by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites. Biochemistry 40, 249–255 (2001).

Farese, R. V. Function and dysfunction of aPKC isoforms for glucose transport in insulin-sensitive and insulin-resistant states. Am. J. Physiol. Endocrinol. Metab. 283, E1–E11 (2002).

Farese, R. V., Sajan, M. P. & Standaert, M. L. Atypical protein kinase C in insulin action and insulin resistance. Biochem. Soc. Trans. 33, 350–353 (2005).

Pouyssegur, J., Volmat, V. & Lenormand, P. Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem. Pharmacol. 64, 755–763 (2002).

Pages, G. et al. Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 286, 1374–1377 (1999).

Bost, F. et al. The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes 54, 402–411 (2005).

Hancock, J. F. & Parton, R. G. Ras plasma membrane signalling platforms. Biochem. J. 389, 1–11 (2005).

Rodriguez-Viciana, P. et al. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370, 527–532 (1994).

Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nature Rev. Mol. Cell Biol. 5, 875–885 (2004).

Matos, P. et al. Small GTPase Rac1: structure, localization, and expression of the human gene. Biochem. Biophys. Res. Commun. 277, 741–751 (2000).

Marks, P. W. & Kwiatkowski, D. J. Genomic organization and chromosomal location of murine Cdc42. Genomics 38, 13–18 (1996).

Marcusohn, J., Isakoff, S. J., Rose, E., Symons, M. & Skolnik, E. Y. The GTP-binding protein Rac does not couple PI 3-kinase to insulin-stimulated glucose transport in adipocytes. Curr. Biol. 5, 1296–1302 (1995).

Usui, I., Imamura, T., Huang, J., Satoh, H. & Olefsky, J. M. Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes. J. Biol. Chem. 278, 13765–13774 (2003).

Ip, Y. T. & Davis, R. J. Signal transduction by the c-Jun N-terminal kinase (JNK) — from inflammation to development. Curr. Opin. Cell Biol. 10, 205–219 (1998).

Zarubin, T. & Han, J. Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15, 11–18 (2005).

Furtado, L. M., Somwar, R., Sweeney, G., Niu, W. & Klip, A. Activation of the glucose transporter GLUT4 by insulin. Biochem. Cell Biol. 80, 569–578 (2002).

Carlson, C. J., Koterski, S., Sciotti, R. J., Poccard, G. B. & Rondinone, C. M. Enhanced basal activation of mitogen-activated protein kinases in adipocytes from type 2 diabetes: potential role of p38 in the downregulation of GLUT4 expression. Diabetes 52, 634–641 (2003).

Chiang, S. H. et al. TCGAP, a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport. EMBO J. 22, 2679–2691 (2003).

Thien, C. B. & Langdon, W. Y. Cbl: many adaptations to regulate protein tyrosine kinases. Nature Rev. Mol. Cell Biol. 2, 294–307 (2001).

Molero, J. C. et al. c-Cbl-deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action. J. Clin. Invest. 114, 1326–1333 (2004).

Zhou, Q. L. et al. Analysis of insulin signalling by RNAi-based gene silencing. Biochem. Soc. Trans. 32, 817–821 (2004).

Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J. & Elledge, S. J. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc. Natl Acad. Sci. USA 102, 13212–13217 (2005).

Shinagawa, T. & Ishii, S. Generation of Ski-knockdown mice by expressing a long double-strand RNA from an RNA polymerase II promoter. Genes Dev. 17, 1340–1345 (2003).

Ding, S. et al. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122, 473–483 (2005).

Becker, A. B. & Roth, R. A. Insulin receptor structure and function in normal and pathological conditions. Annu. Rev. Med. 41, 99–115 (1990).

Goren, H. J., White, M. F. & Kahn, C. R. Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity. Biochemistry 26, 2374–2382 (1987).

Sesti, G. et al. Tissue-specific expression of two alternatively spliced isoforms of the human insulin receptor protein. Acta Diabetol. 31, 59–65 (1994).

Yamaguchi, Y. et al. Functional properties of two naturally occurring isoforms of the human insulin receptor in Chinese hamster ovary cells. Endocrinology 129, 2058–2066 (1991).

Leibiger, B. et al. Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic β cells. Mol. Cell 7, 559–570 (2001).

Bhalla, U. S. & Iyengar, R. Emergent properties of networks of biological signaling pathways. Science 283, 381–387 (1999).


It is often the case that occupied receptors activate protein kinases, which activate other protein kinases, which activate yet other protein kinases to produce phospho-proteins which may act as transcription factors. An example is the mitogen activated protein kinase (MAPK system). A mitogen is an external chemical signal that causes mitosis or cell division. Activated of transcription factors by their phosphorylation through a mitogen activated kinase is required. The sequence of events is:

binding of external signal to membrane receptor and activation of receptor kinase

phosphorylation of receptor kinase and interaction with an activator GTP binding protein like ras

binding of activated G-protein to and activation of a mitogen activated protein kinase kinase kinase (MAPKKK)

MKKK phosphorylates and activates another kinase, MAPKK

MKK phosphorylates and activates mitogen activated protein kinase, MAPK

MAPK phosphorylates inactive transcription factors (or other proteins) and activates them. Unfortunately (from a naming point of view) when the activated proteins are themselves protein kinase, they are called mitogen activated protein kinase activated protein kinases (MAPKAPK)

There are seven types of MAPKs, four conventional and three atypical. Four typical ones are described in the table below.

Activator GTP binding protein Ras:GTP
MAPKKK or MAPK3 Raf-1A/B
c-Mos
MEKK1-4
DLK
MLK2
MEKK1-4
DLK
MLK2
MEKK2/3
Tpl-2
MAPKK or MAPK2 MEK1,2 MEK4,7 MEK3,6 MEK5
MAPK or MAK ERK1,2 JNK1-3 p38 ERK5
MAPKAPK
RSK 1-4
MNK2
MSK 1,2
MK2,3 MSK1,2
MK2,3
RSK1-4
An eventual
Protein Target
c-Jun c-Jun

The Enzymes

Dudley W. Lamming , David M. Sabatini , in The Enzymes , 2010

VI The Regulation of mTOR Signaling by Insulin and PRAS40

Insulin signaling via mTORC1 is positively stimulated by the GTP-binding protein Rheb, which is itself negatively regulated by the action of the tuberous sclerosis tumor suppressor proteins TSC1/TSC2 [ 26 ]. TSC2 is a GTPase-activating protein, and the loss or mutation of TSC2 results in the constitutive loading of Rheb with GTP and the constitutive activation of mTORC1 signaling. The TSC1/2 complex serves as a signal-integration hub for a variety of nutrient-related signaling to mTORC1. Energy deprivation by AMPK, MAPK signaling, Wnt signaling, and hypoxia all regulate mTORC1 signaling via the regulation of TSC1/2 and Rheb [ 27–29 ]. AMPK also regulates mTOR signaling by directly phosphorylating Raptor and inhibiting its binding to mTOR [ 30 ].

TSC1/2 is also regulated by Akt, and as mentioned above the activity of Akt is itself regulated by mTORC2 [ 13, 23 ]. Inhibition of mTORC1 signaling by rapamycin leads to the stabilization of the interaction between IRS1 and the insulin receptor due to a feedback-loop mediated via S6K1 [ 31, 32 ] which in many cell types leads to increased signaling through mTORC2, and as mentioned above the phosphorylation of Ser473 on Akt and its activation [ 13, 32 ]. Akt then acts at three levels to regulate mTORC1 activity. First, it directly phosphorylates TSC2, disrupting the formation of a TSC1/2 complex and thus positively regulating mTORC1 activity [ 33 ]. Secondly, it again potentiates mTORC1 activity by phosphorylating and inhibiting the mTORC1-inhibitor PRAS40 [ 20 ]. Figure 2.2 provides a simplified diagram of the signaling between mTORC1, mTORC2, Akt, and TSC2. Finally, activated Akt stabilizes the surface expression of nutrient transporters, including Glut1 and amino acid transporters, which in turn promotes the uptake of nutrients and activates mTOR signaling [ 34 ].

The role of PRAS40 in the regulation of mTORC1 signaling was, much like many of the other core components of mTORC1, discovered at approximately the same time by different teams of researchers. A mass-spectrometry-based approach was used to examine mTOR immunoprecipitates [ 20, 21 ]. The team of Vander Haar et al. then used a direct approach to discover additional proteins bound to mTOR, while Sancak et al. discovered PRAS40 as a consequence of the development of an in vitro kinase assay for mTORC1. They found that mTORC1 immunoprecipitated from either insulin-stimulated or serum-starved cells was equally active, leading to the hypothesis that perhaps an additional factor that conferred insulin sensitivity was being lost during the purification process. They discovered that washing with low-salt buffers during the immunoprecipitation enabled them to recover complexes that had an insulin-induced activity difference, and subsequently identified the Akt-substrate PRAS40 as a salt-sensitive factor that inhibits mTORC1 during insulin deprivation [ 20 ]. While PRAS40 is an mTORC1 inhibitor, its action can be overcome in vitro by Rheb loaded with GTP, demonstrating that this is likely how insulin signaling to mTORC1 overcomes the effect of PRAS40 in vivo. While mTOR signaling is highly conserved, PRAS40 appears to be a more recent evolutionary development, as it is not found in yeast. However, a Drosophila homologue of PRAS40, Lobe, also functions as an mTORC1 inhibitor, demonstrating that this protein has been an important mTORC1 regulator for a substantial period of evolutionary time [ 20 ].

Subsequent work shed additional light on how PRAS40 functions and its potential clinical relevance. PRAS40 is now believed to function as a director inhibitor of substrate binding to Raptor and may itself also be an mTOR substrate [ 35–37 ]. PRAS40 has been identified as a target of Akt3 activity during malignant melanomas, and phosphorylated PRAS40 is believed to protect cancer cells from apoptosis [ 38 ]. However, this same property of PRAS40 may be beneficial in some contexts, and transfection of PRAS40 protects motor neurons from death in a mouse model of spinal cord injury [ 39 ].


Rapid activation of protein kinase B/Akt has a key role in antiapoptotic signaling during liver regeneration

Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones, and cytokines. Here we report that protein kinase B (PKB)/Akt, part of a key cell survival signaling pathway, is markedly activated after partial hepatectomy (PHX). The antiapoptotic protein Bad, a downstream target of PKB/Akt, is also phosphorylated. This cascade can be activated by various factors in primary hepatocytes, with the strongest activation by insulin and the alpha1-adrenergic agonist phenylephrine (PE), followed by IL-6, epidermal growth factor (EGF), and hepatocyte growth factor (HGF). Pretreatment of cells with the specific PI3 kinase inhibitor LY294002 abolished insulin- or PE-activation of PKB/Akt, suggesting that activation of PKB/Akt is mediated by a PI3 kinase-dependent mechanism. In vivo administration of PE, insulin, IL-6, HGF, or EGF to mice markedly stimulated PKB/Akt in the liver, with the strongest stimulation induced by insulin and PE. Moreover, HGF and insulin were able to attenuate transforming growth factor beta-induced apoptosis in hepatic cells, and these effects were antagonized by LY294002. Taken together, these findings suggest that rapid activation of PKB/Akt is a key antiapoptotic signaling pathway involved in liver regeneration.


Role of PI3K/AKT, cPLA2 and ERK1/2 Signaling Pathways in Insulin Regulation of Vascular Smooth Muscle Cells Proliferation

Author(s): Esma R. Isenovic, Mamdouh H. Kedees, Snezana Tepavcevic, Tijana Milosavljevic, Goran Koricanac, Andreja Trpkovic, Pierre Marche INSERM UMR 956, Faculte de Medecine Pitie-Salpetriere, 91 Bd l'Hopital, 75634 Paris Cedex 13, France., France

Affiliation:

Journal Name: Cardiovascular & Hematological Disorders-Drug Targets
Formerly Current Drug Targets - Cardiovascular & Hematological Disorders

Volume 9 , Issue 3 , 2009




Abstract:

Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.

Cardiovascular & Hematological Disorders-Drug Targets

Title: Role of PI3K/AKT, cPLA2 and ERK1/2 Signaling Pathways in Insulin Regulation of Vascular Smooth Muscle Cells Proliferation

VOLUME: 9 ISSUE: 3

Author(s):Esma R. Isenovic, Mamdouh H. Kedees, Snezana Tepavcevic, Tijana Milosavljevic, Goran Koricanac, Andreja Trpkovic and Pierre Marche

Affiliation:INSERM UMR 956, Faculte de Medecine Pitie-Salpetriere, 91 Bd l'Hopital, 75634 Paris Cedex 13, France.

Abstract: Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.