Lecture 17. Regulation of gene expression by second messengers other than camp. camp cgmp IP3 DAG Ca 2+

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1 Lecture 17 Regulation of gene expression by second messengers other than cam cam cgm I3 DAG Ca 2+

2 Lectures 15 and 16 Introduction to signal transduction Lipophilic signalling molecules and intracellular receptors Water soluble signalling molecules and membrane receptors Activation of adenylate cyclase and synthesis of cam by GCR signalling pathway involving trimeric G proteins Synthesis of cam and activation of protein kinase A Regulation of gene expression by cam and protein kinase A via ATF/CREB family of transcription factors Regulation of gene expression by cgm, I3, DAG, Ca 2+

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4 Epinephrine, Norepinephrine 1 Netrin N Gcg N G c g R C Gα A D R Gα-Q, AC Gα-S AT cam D D C C C C + +Cl K - Ca2+ Ca2+ K C Ca2+ al di ar tion c yo ac M n tr Co N K+ Na + Cl Gα GD Ca2+ N G C R C C N N G G SCn, KCn, ClCn, CaCn Gα-I GT Gγ Gβ Calm Ucn Akt Signaling Gα-S Akt Metabolic Energy Gβ CamK2 Fac Growth tor R ece ptor s I3K Src C Gα-S GD Gro w Fac th tor Ucn2 C R H R Arrβ RGS C sa cam athway Neurotransmitters, Hormones, Stress, Inflammatory Stimuli GT AT Na LC DAG Gγ YK2 HCO 3 Ca2+ I2 KC CamKs Ras Raf1 I3 Caln CamK4 ACI ACII ACVI ACV ACIII ACVII ACIV ACVIII ACIX MEK1, BAD Cell Survival A K A HSL KAR KAR enos YG ln TnnI GSK3 Adducin Oncogenesis GYS Glycogen Synthesis roteinlounge.com roteinlounge.com RhoA Rho Kinase Rap2 athway p38 Signaling Cytokine roduction rotein Retention Ca2+ R y R Ca NFAT Cell Survival AC K D E L Ca2+ R NFAT AC Regulation Cell Survival T Caln tase1 GD BRaf Fatty Acid Metabolism DAR32 Myosin II/ RLC Endothelial Cell Regulation CTK1 Cell roliferation Rap1A Rap2 A cam GEFI p38 LA2 Cardiovascular Homeostasis Regulation of Cytoskeleton p70s6k cam GEFII cam KI enos Signaling DE Mep1B CR Myocardial Contraction C mtor KAC KA KA Glycolysis AM KAC HK Glycogen Glucose-1hosphate AT Metabolic Energy /BAD athway Lipolysis Cell Growth Cell Survival, Chemotaxis mtor Signaling p70s6k athway Rap1 athway ATF1 CREB Degradation of Cell Cycle Regulators ICER CREM CB NF-κB Elk1 Gene Expression Cell Survival Axonal Growth I 3 R ERK

5 KA S199 hnrn-a S44 ATF S133 S191 S216 CREB ER81 ETS 50549

6 cam dependent-protein kinase A pathway Today s lecture cgm-dependent protein kinase G pathway Ca 2+ -CaMK and DAG-protein kinase C pathways

7 peptide hormones neurotransmitters cam cgm AF Ca 2+ neurotransmitters hypothalamic pituitrin G AC GC G L AT GT I 2 (C) I 3 Ca 2+ -CaM KA KG KC CaM-K DG cell memberane phosphorylation of enzymes or proteins effects nucleus trans-acting factor cis-acting element expression

8 Ca 2+ signalling

9 Calcium ions activate Calcium-Calmodulin Kinases (CaM Kinases) which phosphorylate CREB and thus activate genes containing CREs Ca++ enters cell Ca++ activates calmodulin Calmodulin activates CaM kinases CaM kinases phosphorylate multiple targets including ser 133 of CREB

10 Ca 2+ dependent protein kinase pathway (1) Ca 2+ -DAG -dependent KC pathway I 2 H R G protein LC I 3 ER Ca 2+ DG S KC hosphorylation of enzymes / proteins Biological effects

11 (2) Ca 2 -CaM dependent protein kinase pathway H R G protein LC I 3 Ca 2+ CaM CaMK hosphorylation of enzymes or proteins Biological effects

12 Calmodulin (CaMKII) Ca 2+ binding protein 4 Ca 2+ CaM Ca 2+ -CaM CaM kinase Ser/Thr - Ca 2+ pump, AC GC Enzymes (glycogen synthase, phosphorylase kinase)

13 Signalling Molecule Receptor Gs Adenylate Cyclase Signalling Molecule Receptor Gq hospholipase C cam DAG and I3 KA cam ATHWAY Ca 2+ release, Calmodulin Kinases & rotein Kinase C Ca 2+ ATHWAY

14 cgm signalling

15 cgm cgm is produced by Guanylate cyclase which catalyzes the conversion of GM to cyclic GM. The intracellular targets of cgm are: cgm-dependent protein kinases (G-kinases); cgm-regulated phosphodiesterases; cgm-gated ion channels.

16 Second messengers such as calcium and cgm regulate multiple cellular processes

17 Regulation of gene expression by cyclic GM Guanylate cyclase (GC) receptor Membrane receptor Soluble receptor Atrial natriuretic peptide (AN) (induce vascular dilatation) NO, CO Since the focus of this lecture series is primarily on regulation of gene expression, we shall skip the activities of soluble GC receptors How does AN regulate gene expression by cgm pathway?

18 AN binds to its receptor and stimulates its intracellular guanylyl cyclase (GC) domain. The AN receptor has been named natriuretic peptide receptor 1 or guanylyl cyclase A (NR1/GCA), a 130-kDa transmembrane protein that converts GT cgm. The active NR1/GCA receptor is a homodimer containing an extracellular AN-binding domain at its aminoterminal end and an intracellular GC domain at its carboxy-terminal end. cgm molecules thus synthesized, bind to target proteins, including the cgm-dependent protein kinases (KG) I and II, the cyclicnucleotide gated ion channels and the cyclic nucleotide phosphodiesterases (DEs). On activation by cgm, KG phosphorylates proteins such as CREB, ATF-1 which in turn activate the expression of target genes.

19 Transcriptional regulation by cgm can also occur indirectly. Indirect control affects upstream signalling pathways modulating specific targets. For example, cgm activates mitogen-activated protein (MA) kinases which regulate several transcription factors, such as CREB, ternary complex factor, activating transcription factor-2 (ATF-2) and c-jun.

20 cgm-dependent KG pathway AN Receptorlinked GC cgm KG NO, CO Soluble GC hosphorylation of enzymes or functional proteins Biological effects

21 AN GC NO cgm KG Vascular dilatation

22 Intracellular cyclic GM concentrations are regulated by the action of guanylyl cyclases and by the rate of degradation by cyclic GM-specific phosphodiesterases, NO activates soluble guanylyl cyclase, which in turn catalyzes the formation of cyclic GM from GT, whereas cyclic GM-specific phosphodiesterases catalyze the hydrolysis of cyclic GM to GM, ending signal transduction. hosphodiesterase 5, phosphodiesterase 6 and phosphodiesterase 9 are specific for cyclic GM Sildenafil (Viagra ), a novel inhibitor of the cyclic GM-specific phosphodiesterase 5, increases intracellular concentrations of cyclic GM and thus is effective in the clinical management of erectile dysfunction. Terrett et al., Sildenafil (Viagra ), a potent and selective inhibitor of type 5 cgm phosphodiesterase with utility for the treatment of male erectile dysfunction. Bioorg. Med. Chem. Lett. 6 (1996), pp Boolell et al., Sildenafil, a novel effective treatment for male erectile dysfunction. Br. J. Urol. 78 (1996), pp

23 In the central nervous system, the nitric oxide (NO)-sensitive sgc isoform is the major enzyme responsible for cgm synthesis. hosphodiesterases (DEs) are enzymes for hydrolysis of cgm in the brain, and they are mainly isoforms. The NO/cGM signaling pathway has been shown to play an important role in the process underlying learning and memory. Aging is associated with an increase in DE expression and activity and a decrease in cgm concentration.

24 LIID MOLECULES AS SECOND MESSENGERS

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26 Hydrolysis of I2 generates Diacylglycerol (DAG) and Inositol 3 hosphate (I3) DAG together with Ca++ activates rotein Kinase C I3 is involvedin Calcium mobilization

27 Functions of KC regulation of metabolism KC Ser/Thr- Gene expression

28 KC hosphorylation of Transcription factors Activation of genes Cell proliferation

29 I2 derived second messengers hospholipase A2 (LA2) arachidonic acid LC DAG/I3 or ceramide LD phosphatidic acid

30 Cellular effects Mostly through activation of KC Gene expression Cell motility roduction of next generation of second messengers Hormones that act through phospholipid breakdown Epinephrine and norepinephrine (α-adrenergic receptors) Acetylcholine (muscarinic receptor) TNFα

31 I2 breakdown Second messenger system for Gq family of G proteins Gq activates phospholipase C (LC) hospholipase C (LC) is membrane bound enzyme LC β breaks phoshatidylinositol 4,5 bisphosphate (I2) to I3 and DAG I2 breakdown Both I3 and DAG are second messengers I3 increases intracellular calcium levels via the release from intracellular stores such as ER DAG activates protein kinase C (KC)

32 rotein kinase C (KC) signaling Serine/threonine kinase Activated by DAG hosphorylates various cellular effectors Activates transcription factors A-1 (c-fos and cjun - both protooncogenes)

33 Lipid derived second messengers for intercellular signaling Eicosanoids Arachidonic acid derivatives rostaglandins Thromboxanes Leukotrienes Lipoxins Lipid derived second messengers for intercellular signaling Escape the cell and participate in intercellular signaling Sensitize normal physiological responses Important as local inflammatory mediators

34 Membrane Receptor signalling 1. Receptors that regulate cyclic AM formation 2. Receptors that regulate cyclic GM formation 3. Receptors that regulate diacylglycerol (DAG) and inositol triphosphate (I3) 4. Receptors that possess protein kinase activity

35 Regulation of gene expression by rotein Kinase C NEXT CLASS