DOCSLIB.ORG

Inhibition of Glycogen Synthase Kinase 3 Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle Svetlana E

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Inhibition of Glycogen Synthase Kinase 3 Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle Svetlana E Inhibition of Glycogen Synthase Kinase 3 Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle Svetlana E. Nikoulina,1,2 Theodore P. Ciaraldi,1,2 Sunder Mudaliar,1,2 Leslie Carter,1,2 Kirk Johnson,3 and Robert R. Henry1,2 Glycogen synthase kinase (GSK)-3 has been implicated on insulin action, using mechanisms that differ from and in the regulation of multiple cellular physiological pro- are additive to those of insulin. Diabetes 51:2190–2198, cesses in skeletal muscle. Selective cell-permeable re- 2002 versible inhibitors (INHs) of GSK-3 (CT98014 and CHIR98023 [Chiron, Emeryville, CA] and LiCl) were used to evaluate the role of GSK-3 in controlling glucose metabolism. Acute treatment (30 min) of cultured hu- lycogen synthase kinase (GSK)-3 is a serine/ man skeletal muscle cells with either INH resulted in a threonine kinase originally discovered because dose-dependent activation of glycogen synthase (GS) of its ability to phosphorylate and inhibit gly- ϳ ␮ with a maximally effective concentration of 2 mol/l. cogen synthase (GS) (1). Human GSK-3 exists The maximal acute effect of either INH on GS (103 ؎ G as two isoforms, ␣ and ␤, encoded by two distinct genes, 25% stimulation over basal) was greater than the max- -imal insulin response (48 ؎ 9%, P < 0.05 vs. INH); LiCl located on chromosomes 19q13.1-2 and 3q13.3-q21, respec was as effective as insulin. The GSK-3 inhibitor effect, tively (2). GSK-3 is constitutively active in resting cells and like that of insulin, was on the activation state (frac- is inhibited by several hormones such as insulin, endothe- tional velocity [FV]) of GS. Cotreatment of muscle cells lial growth factor, and platelet-derived growth factor. with submaximal doses of INH and insulin resulted in an Insulin has been demonstrated to cause inactivation of .(additive effect on GS FV (103 ؎ 10% stimulation, P < GSK-3, both in vivo (3,4) and in several cell types (5–7 0.05 vs. either agent alone). Glucose incorporation into The mechanism of inactivation is associated with phos- glycogen was also acutely stimulated by INH. While phorylation of the specific serine residues Ser21 and Ser9 prolonged (6–24 h) insulin exposure led to desensitiza- in GSK-3 isoforms ␣ and ␤, respectively. Insulin deactiva- tion of GS, INH continued to activate GS FV for at least tion of GSK-3 is phosphatidylinositol (PI) 3-kinase–depen- 24 h. Insulin and LiCl acutely activated glucose uptake, whereas INH stimulation of glucose uptake required dent and probably occurs through protein kinase B/Akt more prolonged exposure, starting at 6 h and continuing (8,9), properties shared with insulin stimulation of GS. to 24 h. Chronic (4-day) treatment with INH increased GS activity is regulated by allosteric and covalent (phos- .(both basal (154 ؎ 32% of control) and insulin-stimu- phorylation/dephosphorylation) mechanisms (10–12 lated (219 ؎ 74%) glucose uptake. Upregulation of Whereas a number of kinases and phosphatases can act on uptake activity occurred without any change in total GS, GSK-3 plays an important role (13–15). GSK-3 phos- cellular GLUT1 or GLUT4 protein content. Yet the same phorylates GS on three specific residues. This phosphory- chronic treatment resulted in a 65 ؎ 6% decrease in lation causes deactivation of GS and decreases its affinity ؎ GSK-3 protein and a parallel decrease (61 11%) in to allosteric activation by glucose-6-phosphate. In addi- GSK-3 total activity. Together with the INH-induced tion, it has been reported that GSK-3 can also phosphory- increase in insulin-stimulated glucose uptake, there was an ϳ3.5-fold increase (P < 0.05) in insulin receptor late insulin receptor substrate (IRS)-1, a key early substrate (IRS)-1 protein abundance. Despite upregula- molecule in insulin-signaling cascades (16), suggesting the tion of IRS-1, maximal insulin stimulation of Akt phos- potential for involvement of GSK-3 in multiple stages of phorylation was unaltered by INH treatment. The insulin action. results suggest that selective inhibition of GSK-3 has an Lithium ion (Li) has been found to cause relatively impact on both GS and glucose uptake, including effects specific inhibition of GSK-3 (15,17) and has been reported to have insulin-like effects on glucose metabolism, includ- ing increases in glucose uptake, activation of GS activity, From the 1Veterans Affairs San Diego Healthcare System, La Jolla, California; and stimulation of glycogen synthesis in skin, muscle, and the 2Department of Medicine, University of California, San Diego, La Jolla, fat cells. However, Li can also inhibit myo-inositol-1- California; and 3Chiron Corporation, Emeryville, California. Address correspondence and reprint requests to R.R. Henry, MD, VA San monophosphatase (18), 1,6 bisphosphatase, and other Diego Health Care System (9111G), 3350 La Jolla Village Dr., San Diego, CA enzymes involved in regulation of glucose metabolism 92161. E-mail: [email protected]. Received for publication 22 May 2001 and accepted in revised form 27 (19–21). Several other agents have also been reported to March 2002. inhibit GSK-3 activity: Ro 31-8220, valproic acid, iodotu- DTT, dithiothreitol; FV, fractional velocity; GS, glycogen synthase; GSK, bercidin, staurosporin, sangivemycin, and hymenialdisine glycogen synthase kinase; IA, inactive analog; INH, inhibitor; IRS, insulin receptor substrate; MEM, minimal essential medium; PI, phosphatidylinositol; (22). Like lithium, some of these inhibitors have been PMSF, phenylmethylsulfonyl fluoride. reported to exhibit antidiabetic effects (valproate) or to 2190 DIABETES, VOL. 51, JULY 2002 S.E. NIKOULINA AND ASSOCIATES promote glycogen formation (iodotubercidin). However, University of California, San Diego. Informed written consent was obtained these agents are not specific for GSK-3 inhibition and also from all subjects after explanation of the protocol. The method of culturing skeletal muscle cells from biopsy samples has inhibit other protein kinases involved in insulin signal been described in detail in several publications (24,25). Satellite cells were transduction and/or glucose metabolism. Consequently, it obtained by trypsin digestion of muscle biopsy material. Cells were propa- has been difficult to confidently ascribe the insulin-like gated in culture by modifications of the methods described by Blau and effects of these agents to GSK-3 inhibition alone. Webster (28) and Sarabia et al. (29). When cells attained ϳ80% confluency, Several recent observations suggest an important role they were transferred to differentiation media. Differentiation into multinucle- ated myotubes was complete within 4 days. Media were changed every 48 h. for GSK-3 in the regulation of GS and possible involvement When cells were treated chronically for 4 days, media with fresh agents were in the development of insulin resistance in type 2 diabetes. changed every other day. Acutely, cells were treated with the selective GSK-3 Overexpression of GSK-3 in 293 cells and 3T3-L1 adipo- inhibitor CHIR 98024 or its IA in serum-free media for 30 min or as indicated cytes results in decreased GS affinity to glucose-6-phos- and, in some cases, together with insulin (33 nmol/l) at the completion of the phate as well as impaired insulin-stimulated IRS-1 differentiation period. GS activity. GS activity was measured as described in detail previously phosphorylation (15,16). In addition, GSK-3 expression is (25,30). Cells from six-well plates were washed with PBS and scraped into 0.3 elevated in skeletal muscle of type 2 diabetic subjects and ml of extraction buffer (50 mmol/l HEPES, 10 mmol/l EDTA, 100 mmol/l NaF, is inversely related to whole-body insulin action (22). 5 mmol/l dithiothreitol [DTT], 1 ␮mol/l leupeptin, 1 ␮mol/l pepstatin, and 200 However, the physiological role of GSK-3 in insulin-sensi- ␮mol/l PMSF, pH 7.5). Protein concentration was determined by the Bradford tive tissues, such as skeletal muscle and especially its role method (31) with BSA as a standard. GS activity was determined at a in regulation of glucose metabolism, remains to be deter- physiological concentration of substrate (0.3 mmol/l UDP glucose) and expressed in nanomoles of UDP glucose incorporated into glycogen per mined. minute per milligram of total protein and as fractional velocity (FV) (the ratio The current experiments used novel, potent, and selec- of activity assayed at 0.1 mmol/l glucose-6-phosphate divided by the activity at tive inhibitors of GSK-3 to study the regulation of glucose 10 mmol/l glucose-6-phosphate). This is believed to be an indicator of changes metabolism and insulin action in the major insulin target in the phosphorylation state of GS in response to insulin (12). tissue—skeletal muscle. These studies were performed in Glycogen synthesis. Glycogen synthesis was determined in differentiated myotubes as D-[14C]glucose incorporation into glycogen during a 1-h incuba- cultured human skeletal muscle cells, a system that tion at 37°C (24). After incubation, cells were rinsed four times with PBS at closely reflects the in vivo metabolic behavior of skeletal 4°C and solubilized with 1N NaOH at 55°C for 1 h. An aliquot (100 ␮l) of the muscle, including impaired insulin activation of GS (23– lysate was removed for protein analysis. The final glycogen pellets were 25). resuspended in 0.5 ml H2O, mixed with scintillation fluid, and radioactivity determined by liquid scintillation counting. Absolute results are expressed as nanomoles glucose converted to glycogen per milligram protein per hour. Assay of GSK-3 activity. Total GSK-3 (␣ and ␤) activity was measured in the RESEARCH DESIGN AND METHODS homogenates against a peptide substrate (RRAAEELDSRAGpSPQL) derived Materials. UDP-[14C]glucose, D-[U-14C]glucose, L-[14C]glucose, and [3H]-2- from the e-subunit of eIF2-(SP). This peptide and control peptide [where the deoxyglucose were obtained from DuPont NEN (Boston, MA).
Load more

Recommended publications
  • Gene Symbol Gene Description ACVR1B Activin a Receptor, Type IB
    Table S1. Kinase clones included in human kinase cDNA library for yeast two-hybrid screening Gene Symbol Gene Description ACVR1B activin A receptor, type IB ADCK2 aarF domain containing kinase 2 ADCK4 aarF domain containing kinase 4 AGK multiple substrate lipid kinase;MULK AK1 adenylate kinase 1 AK3 adenylate kinase 3 like 1 AK3L1 adenylate kinase 3 ALDH18A1 aldehyde dehydrogenase 18 family, member A1;ALDH18A1 ALK anaplastic lymphoma kinase (Ki-1) ALPK1 alpha-kinase 1 ALPK2 alpha-kinase 2 AMHR2 anti-Mullerian hormone receptor, type II ARAF v-raf murine sarcoma 3611 viral oncogene homolog 1 ARSG arylsulfatase G;ARSG AURKB aurora kinase B AURKC aurora kinase C BCKDK branched chain alpha-ketoacid dehydrogenase kinase BMPR1A bone morphogenetic protein receptor, type IA BMPR2 bone morphogenetic protein receptor, type II (serine/threonine kinase) BRAF v-raf murine sarcoma viral oncogene homolog B1 BRD3 bromodomain containing 3 BRD4 bromodomain containing 4 BTK Bruton agammaglobulinemia tyrosine kinase BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) C9orf98 chromosome 9 open reading frame 98;C9orf98 CABC1 chaperone, ABC1 activity of bc1 complex like (S. pombe) CALM1 calmodulin 1 (phosphorylase kinase, delta) CALM2 calmodulin 2 (phosphorylase kinase, delta) CALM3 calmodulin 3 (phosphorylase kinase, delta) CAMK1 calcium/calmodulin-dependent protein kinase I CAMK2A calcium/calmodulin-dependent protein kinase (CaM kinase) II alpha CAMK2B calcium/calmodulin-dependent
    [Show full text]
  • AGC Kinases in Mtor Signaling, in Mike Hall and Fuyuhiko Tamanoi: the Enzymes, Vol
    Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book, The Enzymes, Vol .27, published by Elsevier, and the attached copy is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research and educational use including without limitation use in instruction at your institution, sending it to specific colleagues who know you, and providing a copy to your institution’s administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: ESTELA JACINTO, AGC Kinases in mTOR Signaling, In Mike Hall and Fuyuhiko Tamanoi: The Enzymes, Vol. 27, Burlington: Academic Press, 2010, pp.101-128. ISBN: 978-0-12-381539-2, © Copyright 2010 Elsevier Inc, Academic Press. Author's personal copy 7 AGC Kinases in mTOR Signaling ESTELA JACINTO Department of Physiology and Biophysics UMDNJ-Robert Wood Johnson Medical School, Piscataway New Jersey, USA I. Abstract The mammalian target of rapamycin (mTOR), a protein kinase with homology to lipid kinases, orchestrates cellular responses to growth and stress signals. Various extracellular and intracellular inputs to mTOR are known. mTOR processes these inputs as part of two mTOR protein com- plexes, mTORC1 or mTORC2. Surprisingly, despite the many cellular functions that are linked to mTOR, there are very few direct mTOR substrates identified to date.
    [Show full text]
  • Constitutive Activation of Akt/Protein Kinase B in Melanoma Leads to Up- Regulation of Nuclear Factor-␬B and Tumor Progression1
    [CANCER RESEARCH 62, 7335–7342, December 15, 2002] Constitutive Activation of Akt/Protein Kinase B in Melanoma Leads to Up- Regulation of Nuclear Factor-␬B and Tumor Progression1 Punita Dhawan, Amar B. Singh, Darrel L. Ellis, and Ann Richmond2 Departments of Veterans Affairs [A. R.], Cancer Biology [P. D., A. R.], Nephrology [A. B. S.], and Medicine, Division of Dermatology [D. L. E.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232 ABSTRACT iting a cumulative melanoma risk of 1.9% compared with 0.04% of patients without dysplastic nevi (7). The current lifetime incidence The serine/threonine kinase Akt/protein kinase B and the pleiotropic of melanoma in the United States is estimated at 1:74 (8). The high transcription factor nuclear factor-␬B [NF-␬B (p50/p65)] play important incidence of dysplastic nevi and melanoma presents a large public roles in the control of cell proliferation, apoptosis, and oncogenesis. Pre- vious studies from our laboratory have shown the constitutive activation health problem. of NF-␬B in melanoma cells. However, the mechanism of this activation is Whereas histopathology is the current gold standard for the diag- not clearly understood. The purpose of this study was to explore the role nosis of atypical melanocytic lesions, interobserver correlation in the of Akt in the activation of NF-␬B during melanoma tumor progression. diagnosis of dysplastic nevi is variable (9, 10). The malignant poten- Based on our observation that two of the five melanoma cell lines exam- tial of dysplastic nevi and early melanomas is difficult to predict with ined exhibit constitutive Akt activation, we evaluated Akt activation by standard histological staining.
    [Show full text]
  • Glycogen Synthase Kinase-3 Is Activated in Neuronal Cells by G 12
    The Journal of Neuroscience, August 15, 2002, 22(16):6863–6875 Glycogen Synthase Kinase-3 Is Activated in Neuronal Cells by G␣ ␣ 12 and G 13 by Rho-Independent and Rho-Dependent Mechanisms C. Laura Sayas, Jesu´ s Avila, and Francisco Wandosell Centro de Biologı´a Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´ noma de Madrid, Cantoblanco, Madrid 28049, Spain ␣ ␣ ␣ ␣ Glycogen synthase kinase-3 (GSK-3) was generally considered tively active G 12 (G 12QL) and G 13 (G 13QL) in Neuro2a cells a constitutively active enzyme, only regulated by inhibition. induces upregulation of GSK-3 activity. Furthermore, overex- Here we describe that GSK-3 is activated by lysophosphatidic pression of constitutively active RhoA (RhoAV14) also activates ␣ acid (LPA) during neurite retraction in rat cerebellar granule GSK-3 However, the activation of GSK-3 by G 13 is blocked by neurons. GSK-3 activation correlates with an increase in GSK-3 coexpression with C3 transferase, whereas C3 does not block ␣ tyrosine phosphorylation. In addition, LPA induces a GSK-3- GSK-3 activation by G 12. Thus, we demonstrate that GSK-3 is ␣ ␣ mediated hyperphosphorylation of the microtubule-associated activated by both G 12 and G 13 in neuronal cells. However, ␣ protein tau. Inhibition of GSK-3 by lithium partially blocks neu- GSK-3 activation by G 13 is Rho-mediated, whereas GSK-3 ␣ rite retraction, indicating that GSK-3 activation is important but activation by G 12 is Rho-independent. The results presented not essential for the neurite retraction progress. GSK-3 activa- here imply the existence of a previously unknown mechanism of ␣ tion by LPA in cerebellar granule neurons is neither downstream GSK-3 activation by G 12/13 subunits.
    [Show full text]
  • PRKCQ Promotes Oncogenic Growth and Anoikis Resistance of a Subset
    Byerly et al. Breast Cancer Research (2016) 18:95 DOI 10.1186/s13058-016-0749-6 RESEARCH ARTICLE Open Access PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple- negative breast cancer cells Jessica Byerly1†, Gwyneth Halstead-Nussloch1†, Koichi Ito1, Igor Katsyv3 and Hanna Y. Irie1,2* Abstract Background: The protein kinase C (PKC) family comprises distinct classes of proteins, many of which are implicated in diverse cellular functions. Protein tyrosine kinase C theta isoform (PRKCQ)/PKCθ, a member of the novel PKC family, may have a distinct isoform-specific role in breast cancer. PKCθ is preferentially expressed in triple-negative breast cancer (TNBC) compared to other breast tumor subtypes. We hypothesized that PRKCQ/PKCθ critically regulates growth and survival of a subset of TNBC cells. Methods: To elucidate the role of PRKCQ/PKCθ in regulating growth and anoikis resistance, we used both gain and loss of function to modulate expression of PRKCQ. We enhanced expression of PKCθ (kinase-active or inactive) in non-transformed breast epithelial cells (MCF-10A) and assessed effects on epidermal growth factor (EGF)- independent growth, anoikis, and migration. We downregulated expression of PKCθ in TNBC cells, and determined effects on in vitro and in vivo growth and survival. TNBC cells were also treated with a small molecule inhibitor to assess requirement for PKCθ kinase activity in the growth of TNBC cells. Results: PRKCQ/PKCθ can promote oncogenic phenotypes when expressed in non-transformed MCF-10A mammary epithelial cells; PRKCQ/PKCθ enhances anchorage-independent survival, growth-factor-independent proliferation, and migration. PKCθ expression promotes retinoblastoma (Rb) phosphorylation and cell-cycle progression under growth factor-deprived conditions that typically induce cell-cycle arrest of MCF-10A breast epithelial cells.
    [Show full text]
  • Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids
    Supplementary Table 1. In vitro side effect profiling study for LDN/OSU-0212320. Percent Inhibition Receptor 10 µM Neurotransmitter Related Adenosine, Non-selective 7.29% Adrenergic, Alpha 1, Non-selective 24.98% Adrenergic, Alpha 2, Non-selective 27.18% Adrenergic, Beta, Non-selective -20.94% Dopamine Transporter 8.69% Dopamine, D1 (h) 8.48% Dopamine, D2s (h) 4.06% GABA A, Agonist Site -16.15% GABA A, BDZ, alpha 1 site 12.73% GABA-B 13.60% Glutamate, AMPA Site (Ionotropic) 12.06% Glutamate, Kainate Site (Ionotropic) -1.03% Glutamate, NMDA Agonist Site (Ionotropic) 0.12% Glutamate, NMDA, Glycine (Stry-insens Site) 9.84% (Ionotropic) Glycine, Strychnine-sensitive 0.99% Histamine, H1 -5.54% Histamine, H2 16.54% Histamine, H3 4.80% Melatonin, Non-selective -5.54% Muscarinic, M1 (hr) -1.88% Muscarinic, M2 (h) 0.82% Muscarinic, Non-selective, Central 29.04% Muscarinic, Non-selective, Peripheral 0.29% Nicotinic, Neuronal (-BnTx insensitive) 7.85% Norepinephrine Transporter 2.87% Opioid, Non-selective -0.09% Opioid, Orphanin, ORL1 (h) 11.55% Serotonin Transporter -3.02% Serotonin, Non-selective 26.33% Sigma, Non-Selective 10.19% Steroids Estrogen 11.16% 1 Percent Inhibition Receptor 10 µM Testosterone (cytosolic) (h) 12.50% Ion Channels Calcium Channel, Type L (Dihydropyridine Site) 43.18% Calcium Channel, Type N 4.15% Potassium Channel, ATP-Sensitive -4.05% Potassium Channel, Ca2+ Act., VI 17.80% Potassium Channel, I(Kr) (hERG) (h) -6.44% Sodium, Site 2 -0.39% Second Messengers Nitric Oxide, NOS (Neuronal-Binding) -17.09% Prostaglandins Leukotriene,
    [Show full text]
  • Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoform
    Molecular Pharmacology Fast Forward. Published on November 25, 2020 as DOI: 10.1124/molpharm.120.000094 This article has not been copyedited and formatted. The final version may differ from this version. Distinct regulation of cardiac fibroblast proliferation and transdifferentiation by classical and novel protein kinase C isoforms: possible implications for new antifibrotic therapies S. Tuuli Karhu, Heikki Ruskoaho, Virpi Talman* Affiliations Downloaded from STK, HR, and VT: Drug Research Program and Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Finland molpharm.aspetjournals.org *Corresponding author at ASPET Journals on September 30, 2021 1 Molecular Pharmacology Fast Forward. Published on November 25, 2020 as DOI: 10.1124/molpharm.120.000094 This article has not been copyedited and formatted. The final version may differ from this version. Running title: PKC agonists inhibit cardiac fibroblast activation Corresponding author: Virpi Talman Division of Pharmacology and Pharmacotherapy Faculty of Pharmacy University of Helsinki P.O. Box 56 (Viikinkaari 5E) FI-00014 Helsinki, FINLAND Downloaded from Tel: +358504480768 Email: [email protected] molpharm.aspetjournals.org Number of text pages: 35 Number of tables: 0 Number of figures: 4 at ASPET Journals on September 30, 2021 Number of references: 55 Number of words in the Abstract: 249 Number of words in the Introduction: 728 Number of words in the Discussion: 1459 Abbreviations: α-SMA, α-smooth muscle actin; aPKC, atypical protein kinase C isoform; BrdU, 5-bromo-2’-deoxyuridine; CF, cardiac fibroblast; cPKC, classical protein kinase C isoform; DDR2, discoidin domain receptor 2; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; HCA, high-content analysis; LDH, lactate dehydrogenase; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nPKC, novel protein kinase C isoform; PKC, protein kinase C 2 Molecular Pharmacology Fast Forward.
    [Show full text]
  • Protein Kinase C As a Paradigm Alexandra C
    Biochem. J. (2003) 370, 361–371 (Printed in Great Britain) 361 REVIEW ARTICLE Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm Alexandra C. NEWTON1 Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0640, U.S.A. Phosphorylation plays a central role in regulating the activation down-regulation of PKC as a paradigm for how these sites and signalling lifetime of protein kinases A, B (also known as control the function of the ABC kinases. Akt) and C. These kinases share three conserved phosphorylation motifs: the activation loop segment, the turn motif and the Key words: phosphoinositide 3-kinase, phosphoinositide-depen- hydrophobic motif. This review focuses on how phosphorylation dent kinase-1 (PDK-1), phosphorylation motif, protein kinase A at each of these sites regulates the maturation, signalling and (PKA), protein kinase B (PKB)\Akt, protein kinase C (PKC). INTRODUCTION by phosphorylation as a paradigm for control of ABC kinase function by phosphorylation. Almost half a century ago Krebs, Graves and Fischer reported b that the first discovered protein kinase, phosphorylase kinase, ARCHITECTURE OF ABC KINASES was, itself, activated by phosphorylation [1]. Two decades later, Fischer and colleagues showed that the kinase that phosphory- Protein kinases A, B\Akt and C have in common a conserved lates phosphorylase kinase, later named protein kinase A (PKA), kinase core whose function is regulated allosterically by a was also phosphorylated [2]. Reversible control by phosphory- corresponding regulatory moiety (Figure 1). In the case of PKA, lation\dephosphorylation is now firmly established as a fun- the regulatory moiety is a separate polypeptide, whereas the damental mechanism for regulating the function of most of the regulatory determinants are on the same polypeptide as the 500 or so members of the mammalian protein kinase superfamily.
    [Show full text]
  • Interaction Between the Protein Kinase B-Raf and the A-Subunit of the IIS Proteasome Regulator1
    (CANCER RESEARCH 58. 2986-2990, July 15, I998| Advances in Brief Interaction between the Protein Kinase B-Raf and the a-Subunit of the IIS Proteasome Regulator1 Andreas Kalmes,2 Garsten Hagemann,2 Christoph K. Weber, Ludmilla Wixler, Tillman Schuster, and Ulf R. Kapp* InstituífürMedizinische Strahlenkunde und Zettforschung (MSZ). University of Würzburg,97078 Würzburg,Germany ¡C.H.. C. K. W.. L. W.. T. S.. U. K. R.], and Department of Surgery, University of Washington, Seattle. Washington 9SI95 ¡A.KJ Abstract using the yeast two-hybrid system. In addition to the already known B-Raf-binding proteins (MEK and several 14-3-3 isoforms), we iso Protein kinases of the Raf family act as signal-transducing elements lated PA28a, the subunit of the 1IS proteasome regulator (11, 12), as downstream of activated cell surface receptors and are involved in the a novel B-Raf-binding partner. regulation of proliferation, differentiation, and cell survival. Whereas the role of c-Raf-1 as a mitogen-activated protein/extracellular signal-regu The 11S regulator is one of two known activators of the 20S lated kinase activator within the mitogenic cascade is well established, less proteasome (13). Both of these activators seem to possess different is known about the mammalian Raf isoforins A-Raf and B-Raf. Here we functions in vivo. The PA700 activator complex (19S regulator) binds report that B-Raf binds to PA28a, one of two subunits of the 1IS regulator to the 20S proteasome core complex to form the active 26S protea of proteasomes. PA28<* was isolated as a B-Raf-binding protein in a yeast some, which mediates the ATP- and ubiquitination-dependent degra two-hybrid screen of a PC 12 cDNA library.
    [Show full text]
  • Protein Kinase C As a Therapeutic Target in Non-Small Cell Lung Cancer
    International Journal of Molecular Sciences Review Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer Mohammad Mojtaba Sadeghi 1,2, Mohamed F. Salama 2,3,4 and Yusuf A. Hannun 1,2,3,* 1 Department of Biochemistry, Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA; [email protected] 2 Stony Brook Cancer Center, Stony Brook University Hospital, Stony Brook, NY 11794, USA; [email protected] 3 Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA 4 Department of Biochemistry, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Dakahlia Governorate, Egypt * Correspondence: [email protected] Abstract: Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. Citation: Sadeghi, M.M.; Salama, PKC isoforms α, ", η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates M.F.; Hannun, Y.A. Protein Kinase C with worse prognosis in NSCLC patients.
    [Show full text]
  • Chem331 Glycogen Metabolism
    Glycogen metabolism Glycogen review - 1,4 and 1,6 α-glycosidic links ~ every 10 sugars are branched - open helix with many non-reducing ends. Effective storage of glucose Glucose storage Liver glycogen 4.0% 72 g Muscle glycogen 0.7% 245 g Blood Glucose 0.1% 10 g Large amount of water associated with glycogen - 0.5% of total weight Glycogen stored in granules in cytosol w/proteins for synthesis, degradation and control There are very different means of control of glycogen metabolism between liver and muscle Glycogen biosynthetic and degradative cycle Two different pathways - which do not share enzymes like glycolysis and gluconeogenesis glucose -> glycogen glycogenesis - biosynthetic glycogen -> glucose 1-P glycogenolysis - breakdown Evidence for two paths - Patients lacking phosphorylase can still synthesize glycogen - hormonal regulation of both directions Glycogenolysis (glycogen breakdown)- Glycogen Phosphorylase glycogen (n) + Pi -> glucose 1-p + glycogen (n-1) • Enzyme binds and cleaves glycogen into monomers at the end of the polymer (reducing ends of glycogen) • Dimmer interacting at the N-terminus. • rate limiting - controlled step in glycogen breakdown • glycogen phosphorylase - cleavage of 1,4 α glycosidic bond by Pi NOT H2O • Energy of phosphorolysis vs. hydrolysis -low standard state free energy change -transfer potential -driven by Pi concentration -Hydrolysis would require additional step s/ cost of ATP - Think of the difference between adding a phosphate group with hydrolysis • phosphorylation locks glucose in cell (imp. for muscle) • Phosphorylase binds glycogen at storage site and the catalytic site is 4 to 5 glucose residues away from the catalytic site. • Phosphorylase removes 1 residue at a time from glycogen until 4 glucose residues away on either side of 1,6 branch point – stericaly hindered by glycogen storage site • Cleaves without releasing at storage site • general acid/base catalysts • Inorganic phosphate attacks the terminal glucose residue passing through an oxonium ion intermediate.
    [Show full text]
  • Dynamic Modelling of the Mtor Signalling Network Reveals Complex
    www.nature.com/scientificreports OPEN Dynamic modelling of the mTOR signalling network reveals complex emergent behaviours conferred by Received: 24 August 2017 Accepted: 1 December 2017 DEPTOR Published: xx xx xxxx Thawfeek M. Varusai1,4 & Lan K. Nguyen2,3,4 The mechanistic Target of Rapamycin (mTOR) signalling network is an evolutionarily conserved network that controls key cellular processes, including cell growth and metabolism. Consisting of the major kinase complexes mTOR Complex 1 and 2 (mTORC1/2), the mTOR network harbours complex interactions and feedback loops. The DEP domain-containing mTOR-interacting protein (DEPTOR) was recently identifed as an endogenous inhibitor of both mTORC1 and 2 through direct interactions, and is in turn degraded by mTORC1/2, adding an extra layer of complexity to the mTOR network. Yet, the dynamic properties of the DEPTOR-mTOR network and the roles of DEPTOR in coordinating mTORC1/2 activation dynamics have not been characterised. Using computational modelling, systems analysis and dynamic simulations we show that DEPTOR confers remarkably rich and complex dynamic behaviours to mTOR signalling, including abrupt, bistable switches, oscillations and co-existing bistable/oscillatory responses. Transitions between these distinct modes of behaviour are enabled by modulating DEPTOR expression alone. We characterise the governing conditions for the observed dynamics by elucidating the network in its vast multi-dimensional parameter space, and develop strategies to identify core network design motifs underlying these dynamics. Our fndings provide new systems-level insights into the complexity of mTOR signalling contributed by DEPTOR. Discovered in the early 1990s as an anti-fungal agent produced by the soil bacterium Streptomyces hygroscopicus, rapamycin has continually surprised scientists with its diverse clinical efects including potent immunosuppres- sive and anti-tumorigenic properties1–3.
    [Show full text]