Revised curriculum (1) December 16 (Tuesday) Second messengers (2) December 18 (Thursday) Protein kinases and cancers (3) December 23 (Tuesday) Protein phosphatases and diseases (4) December 25 (Thursday) Literature study I (5) December 30 (Tuesday) Literature study II Tzu-Ching Meng tcmeng@gate.sinica.edu.tw 27855696 ext 6140 or 6141

Assigned reading for literature study I

Assigned reading for literature study II

Basic concept of cell signaling and second messengers Lecture I Basic concept of cell signaling and second messengers Reference: Lodish et al., Molecular Cell Biology 6th edition (2007), Chapters 15 and 16 December 16, 2008

Cell as information processing unit Unit A Unit B Unit C signals System System Unit Signal society person language computer circuit electrical organism cell chemical Cell Input signal Output signal

General principles of signaling by cell-surface receptors Synthesis and (2) release of signaling molecules by the signaling cells; (3) Transport of the signal to the target cells; (4) Binding of the signal by a specific receptor protein; (5) Initial of intracellular signal transduction pathways; (6) Specific changes in cellular functions; (7) Inactivation of the receptor; (8) Removal of signaling molecules

Signaling molecules can act locally or at a distance

Two modes of intracellular signaling transmission Low molecular weight messenger substances– second messengers Protein-protein interaction Overview of major classes of cell surface receptors 2nd messengers

Relationship between ligand and receptor— G protein coupled receptors

General structure of G protein coupled receptor

Operational model for ligand-induced activation of effector protein associated with G protein coupled receptors

Overview of major classes of cell surface receptors Protein-protein interaction

Relationship between ligand and receptor— Receptors that activate intracellular enzymes

Overview of major classes of cell surface receptors Protein-protein interaction Proteolytic process

Relationship between ligand and receptor— Receptors that trigger intracellular proteolysis pathways

Relationship between ligand and receptor— Receptors that function as ion channel upon ligand binding

A distinct class of receptors— Receptors that are located intracellularly, but ligands may be generated either extracellularly or intracellularly

Characteristics of second messengers: Diffusible signaling molecules; Rapidly produced/activated and rapidly degraded/inactivated; May be stored intracellular in certain organelles; Can be produced in a location-specific manner. Two types of second messengers: Hydrophilic– cAMP, cGMP, inositol phosphates (IPs), Ca2+, NO; Hydrophobic– diacylglycerol (DAG), phosphatidyl inositol phosphates (PIPs).

Four common 2nd messengers

Mammalian adenylyl cyclase is a membrane-bound enzyme with two catalytic domains and two integral membrane domains, each of which contains 6 transmembrane a-helices.

3-D structure of Gsa-GTP complexed with two catalytic domains of adenylyl cyclase One subunit of trimeric G protein

Hormone-induced activation or inhibition of adenylyl cyclase in adipose cells Gs stimulation Gi inhibition

Typical amplification of an external signal downstream from a receptor Extracellular stimulus Effector Intracellular 2nd messenger Effector

The membrane A kinase-associated protein (mAKAP) anchors both PKA and cAMP phosphodiesterase (PDE) to the nuclear membrane, forming a feedback loop that provides close local control of the cAMP level Basal level of PDE activity keeps [cAMP] low; Activation of receptor causes an increase in [cAMP]binding of cAMP to PKA; Activated PKA (C) phosphorylates and activates PDE, driving [cAMP] low; Dephosphorylation of PDE returns the complex to the resting state.

Synthesis of DAG and IP3 from membrane-bound phosphatidylinositol Key enzyme 1 Key enzyme 2 Key enzyme 3 All membrane-bound

IP3/DAG pathway triggered by ligand binding to GPCRs for elevation of Cytosolic Ca2+ and activation of PKC

Second messenger nitric oxide (NO) is produced by intracellular NO Syntheases (NOSs) NADP NADPH nNOS (NOS1) L-citrulline iNOS (NOS2) L-arginine eNOS (NOS3) O2 NO. NO synthases 在細胞中存在三種NOS isoforms, 主要由第一次被發現的位置或者是本身活性上的特性來命名. 分為nNOS, 最初是在neuron cell中發現,也稱為NOSI,另一個為INOS,i為inducible的意思,也稱為NOSII,第三類則是eNOS,是在endothelial cell中被發現,也稱作NOSIII.這3種NOS isoforms目前已知存在在許多的tissue或cell type中. NOS enzyme執行的功能是藉由催化L-arginine以及在其它cofactor的幫助之下,例如NADPH.O2,將L-arginine轉化成L-citrulline以及NO.最初NO的產生被認為是在bacteria中可以發生,後來在mammalian係統中可藉由NOS來產生NO,因此也突顯了NO的重要性. NO is generated in most cell types by NO synthases. nNOS: neuronal NOS iNOS: inducible NOS eNOS: endothelial NOS

Guanylyl cyclase is a typical intracellular receptor of NO NOS NO/cGMP- dependent pathways L-arginine NO + citrulline guanylyl cyclase GTP cGMP cGMP dependent的方式是NO與guanylyl cyclase heme group上的鐵離子作用,造成結構上的改變而活化gyanylyl cyclase,具活性的gyanylyl cyclase可以將GTP催化產生cGMP, cGMP在分別由以下三種不同路徑,phosphodiesterases(PDE)以及 protein kinase G還有一些ion channel的影響來調控cellular effects. 我們來看一下cGMP所影響到的層面有哪些. 在這裡我們可以看到,S-nitrosylation是對蛋白上的cysteine接上NO group的修飾作用,另外NO也可以對蛋白進行其他種形式的修飾,例如在蛋白上的tyrosine接上NO2 group修飾,稱為nitration.目前的了解,S-nitrosylation是在細胞中確實可以進行的蛋白修飾,而對於nitration這樣的蛋白修飾還有所保留.而我們的研究主要就是針對S-nitrosylation進行探討, cGMP-regulated Phosphodiesterases (PDE) Protein kinase G cGMP-regulated Ion channels Smooth muscle relaxation, platelet inhibition , cell growth and differentiation

Regulation of contractility of arterial smooth muscle by NO and cGMP

ROS= Reactive Oxygen Species

Reactive oxygen species (ROS) function as second messengers in cells UV, Ionizing Irradiation Peptide Growth Factors Ligands for GPCRs Cytokines H2O2 Nox ROS ROS ROS ? Activation of Tyr(P) signaling Transformation Signaling Mitogenic Signaling

Assigned paper for the Literature Study I Huang et al (2007) Positive regulation of Itk PH domain function by soluble IP4. Science, 316, p. 886 Reference article: Irvine R. (2007) Cell signaling: the art of the soluble. Science, 316, p. 845