Cellular Signaling 2.pdf

Cell Signaling Part 2 Presented by Dr. Kherie Rowe Assistant Professor of Biochemistry [email protected] 1 There is no required reading. Recommended is Chapter 15 of “Molecular Biology o...

Cell Signaling Part 2 Presented by Dr. Kherie Rowe Assistant Professor of Biochemistry [email protected] 1 There is no required reading. Recommended is Chapter 15 of “Molecular Biology of the Cell” by Alberts et al.. This book is not on our reading list but is a very good general book on cell biology. 1  Learning Objectives 1. Describe the sequence of events in the IP3-DAG cascade 2. Describe the role and effects of calcium as a second messenger 3. Describe the function of nitric oxide in the vascular system 4. Identify the sequence of events in signaling by insulin and growth factor receptors 5. Match insulin, growth factors, cAMP, cGMP, calcium and 2,3- diacylglycerol with the protein kinases that they activate. 2 2  Review from Cell Signaling I Types of Hormones: hydrophobic vs hydrophilic Types of Signaling: Autocrine, paracrine, endocrine, synaptic, contact. Nuclear Hormone receptor signaling (hydrophobic ligands) Receptor theory: Ligand binding, agonists, antagonists, etc. GPCRs and G-protein mediated signaling cascades Concept of second messengers 3 3  Coverage In Cell Signaling 1: – General signaling mechanisms – Nuclear receptor mediated signaling – G-protein mediated signaling Continuation with: – Ca2+ second messenger mediated signaling – Receptor Tyrosine Kinase (RTK) Signaling – Example Pathways and integration 4 4 Role of GPCRs in the autonomic nervous system GPCRs are key in nervous system regulation of autonomic function Acetylcholine via muscarinic receptors Epinephrine and norepinephrine via a- adrenergic and b-adrenergic receptors 5 5  GPCRs as Drug Targets Largest, single class of drug targets in the genome 134 GPCRs are drug targets More than 73 broadly approved drugs Estimated 29-38 % of all drugs target GPCRs Reference: Sriram and Insel Mol. Pharm, DOI: 10.1124/mol.117.111062 6 6 CALCIUM-MEDIATED SIGNALING PATHWAYS 7 7  Ca2+ Second Messenger Signaling Calmodulin mediates many Ca2+ effects. – Small calcium binding protein – Binds 4 calciums – Binding radically alters the structure – Calcium binding causes binding of Calmodulin to target proteins and affects their activity – Calmodulin is homologous to many other calcium binding proteins: Troponin C Parvalbumin S100B 8 Calcium As a Second Messenger cAMP is considered a second messenger: A second messenger is secondary in events to the primary messenger. The primary messenger is typically a hormone that binds externally. Ca2+ is another such second messenger. 1. Calcium is present in low concentrations in the cell (about 100 nM) and high concentration outside (about 2 mM). 1. One reason for this arrangement is that Ca2+ will precipitate phosphate, so because intracellular phosphate can be in the mM range, phosphate and Ca2+ must be segregated. 2. This arrangement also permits large intracellular concentration changes to be effected rapidly, through release of intracellular stores or opening of plasma membrane calcium channels. 2. Calmodulin is one mediator of calcium signaling: 1. Calmodulin binds 4 Ca2+ ions cooperatively. 2. Ca2+ binding changes the calmodulin structure dramatically 3. Ca2+ bound calmodulin can bind to protein targets with a correct calmodulin recognition sequence 4. This typically alters the target protein function dramatically 3. Calmodulin is one of a class of proteins that mediate various aspects of Ca2+ 8  signaling. Others include: 1. Troponin C 2. Parvalbumin (stores Ca2+) 3. S100B 1. Calmodulin modulates the function of many proteins including: 1. CaM Kinase (Calmodulin dependent kinase) which phosphorylates various protein targets in response to Calmodulin binding (see following slides). 2. Ryanodine receptors (calcium activated calcium release channels in sarcoplasmic reticulum) 3. N-type calcium channels 4. Myosin light chain kinase (see later slide). 8  Intracellular Calcium Mobilization Activated by: Calcium-Mediated Effects: – Electrical signaling: – Secretion – mediates fusion of - Calcium Channels vesicles – GPCR mediated signaling – Muscle contraction Stored in endoplasmic reticulum – Muscle relaxation via NO Stored in Sarcoplasmic reticulum – Cell Motility – Hormone synthesis – Glycogen degradation 9 9  GPCR Mediated Ca2+ Mobilization Epinephrine Acetylcholine a1 M1adrenergic Muscarinic receptor 10 Ca2+ signaling activated by hormonal signaling: 1) GPRC (e.g. epinephrine acting on alpha 1-adrenergic receptor or Acetylcholine on a Muscarinic M1 receptor) activates a Gq 2) Gq-GTP activates the effector, phospholipase C-beta 3) Phospholipase C-beta cleaves IP3 from PIP2 4) IP3 acts as a second messenger – It is a ligand for the IP3-receptor and activates it. 5) The IP3 receptor is an internal, ER membrane bound, calcium channel 1) Note – the IP3 receptor is a very large protein 2) The IP3 Receptor opens in response to IP3 3) The IP3 receptor can also open in response to increases in cytosolic Ca2+ 4) Thus, the IP3 receptor can re-inforce its own opening. 5) The IP3 receptor shares homology with the ryanodine receptor – the calcium activated calcium release channel in the sarcoplasmic reticulum of striated muscle. 6) Ca acts as a second messenger and causes downstream effects. Some 2+ examples: 10  1) Smooth muscle contraction. 2) Cell motility 3) Neurotransmitter release 4) Synaptic strengthening 5) Secretion. 1) DiAcylGlycerol, from PIP2 cleavage and Ca2+ from internal stores activate Protein kinase C 1) Protein Kinase C (PKC) has many downstream substrates that it phosphorylates. 2) Both Ca2+ and DAG are required for its activation. 10  Smooth Muscle Contraction: Ca2+ Signaling Ca2+ levels increase: – via IP3, or – Via Ca2+ channels Ca2+ binds calmodulin Ca2+-calmodulin binds MLC kinase Myosin light chains are phosphorylated Contraction occurs 11 Smooth muscle contraction: 1. Contraction is mediated by calcium signaling. 2. Smooth muscle contraction is important in several organs – vascular tone, bronchiae, uterus, etc. 3. Contraction is mediated by changes in calcium concentration. 1. These can be initiated hormonally via GPCRs or through other mechanisms such as channels and electrical signaling. 2. Note – many things can regulate smooth muscle contraction and relaxation – this is not a complete description. Key is the central role of calcium in the process. 4. Contraction occurs through Ca2+ binding to calmodulin (calmodulin is often abbreviated as ‘CaM’) 5. Activated calmodulin then binds directly to Myosin Light Chain Kinase (MLC- kinase) 6. Myosin Light Chain Kinase phosphorylates the myosin light chain. Myosin is part of the contractile apparatus and the phosphorylation causes contraction. 11  Calcium-induced Calcium Release Calcium release can be activated transiently by increased Calcium – Effect is self-desensitizing – Is initiated by: IP3 Calcium entry from channels – Causes waves or oscillations 12 Calcium-activated calcium release: 1. Internal stores of calcium in the ER are released through IP3 receptors or from the sarcoplasmic reticulum through ryanodine receptors (respectively). 2. IP3 receptors and ryanodine receptors are subject to complex regulation, including regulation by calcium itself. 3. Increased calcium concentrations can increase and induce additional calcium release. 4. This effect can cause IP3 receptors to release calcium, and thereby induce further release in nearby channels. 1. This can be seen as calcium waves, or oscillations in calcium concentration. 12 NITRIC OXIDE MEDIATED SIGNALING 13 13  NO Signaling 14 Nitric Oxide ( NO ) This is a small molecule that acts as a smooth muscle relaxant, particularly in the vasculature. It is a free radical so it is a short-lived molecule and acts principally in a paracrine fashion. Furchgott and Murad won the Nobel prize for the discovery of NO as a signaling molecule. Mechanism: 1. Acetylcholine binds to muscarinic acetylcholine receptors (GPCRs) and causes IP3 release and an increase in calcium. This occurs in endothelial cells in the blood lumen. 2. Increased calcium activates NO synthase (NOS). NO is a small molecule, a gas, and a free radical. It diffuses rapidly across membranes and acts in nearby cells. 3. In the surrounding smooth muscle cells, NO binds to guanylate cyclase and activates it. This causes synthesis of cGMP and subsequent activation of cGMP-dependent protein kinases (PKG). 4. The action of cGMP results in muscle relaxation (intervening steps are omitted). 14 NO can be produced by small molecules such as amyl nitrate and nitroglycerin, which is used to relieve angina in patients with coronary artery disease. Such drugs work by rapidly releasing NO and causing vasodilation. NO was once called EDRF for Endothelial derived relaxation factor. 14  NO Signaling ANF and Smooth Muscle Relaxation: Viagra 15 cGMP as a second messenger 1) cGMP can be made by a receptor-enzyme. 1) This enzyme is activated on the cell surface by Atrial Naturetic factor. 2) cGMP can be made by soluble guanylate cyclase, which is activated by NO. 3) cGMP activates Protein Kinase G, and causes vasodilation. 4) cGMP is subsequently degraded by cGMP phosphodiesterases. Action of ANF ANF – atrial natriuretic factor – also an important vasodilator. ANF or ANP (atrial natriuretic peptide) are the same. The ANF receptor in this case does not fall into the class of GPCRs nor ion channels. It is an enzyme linked receptor: 1. ANF binding, extracellularly, activates intracellular Guanylate cyclase activity. 2. cGMP levels rise 3. PKG is activated 4. Smooth muscle relaxes. 5. The signal is turned off by degradation of cGMP phosphodiesterases. 15 Clinical Note: Sildenafil (Viagra) works to treat pulmonary artery hypertension and erectile dysfunction by inhibiting phosphodiesterase 5 (PDE5), keeping cGMP elevated and causing increased relaxation. 15  NO Signaling Effects of Various Vasodilators And Constrictors 16 Vascular tone is controlled by vasodilators and vasoconstrictors. Vasodilators include ANF, acetylcholine, epinephrine, and bradykinin. Vasoconstrictors, angiotensin, vasopressin, epinephrine. Note that alpha and beta-adrenergic receptors have opposing effects. Some vasodilators work directly on smooth muscle and others work through NO release from endothelial cells. 16 RECEPTOR TYROSINE KINASE SIGNALING PATHWAYS 17 17  Growth Factor Receptor Tyrosine Kinases Large family of receptors Contain tyrosine kinase activity Work, typically, by dimerization Many act as mitogen receptors and growth factor receptors 18 Tyrosine Kinase receptors (or Receptor tyrosine kinases, RTKs) General Properties: 1. Single-span transmembrane proteins. 2. Often dimerize as part of the mechanism of action. 3. Contain extracellular ligand binding domains. 4. Contain intracellular tyrosine kinase activity 1. Inactive kinase until ligand bound 2. Ligand binding activates the kinases. 5. Typically undergo auto-phosphorylation – i.e., the tyrosine kinase phosphorylates itself or the paired (dimerized) subunit. 6. The tyrosine phosphorylation acts as a signal for other proteins to bind and propagate activity. 18  RTKs: Receptor Tyrosine Kinases 19 Typical mechanism of action for many RTKs: 1. Inactive RTKs are monomeric 2. Ligand binds, causing dimerization. 3. Dimerization provides a substrate for the tyrosine kinase activity and activates the kinase. 4. Phosphorylated tyrosines and surrounding sequences act as recognition sites for other signaling proteins. Binding activates them and localizes them. 19  RTK Activation of The Small G-protein RAS 20 Small G-proteins: Ras Many RTKs work by activating small G-proteins such as Ras. (Ras is also an oncogene – there are mutations that render it constitutively active causing prolonged mitogenic effects.) Steps in Ras activation: 1. RTK is activated by ligand binding and autophosphorylates (eg. Epidermal growth factor (EGF) receptor) 2. Scaffolding or adaptor proteins bind to the phosphotyrosines. This binding requires a specific domain, an SH2 domain (sarc homology domain 2). 3. The next protein to bind is Sos, a Ras Guanosine Exchange Factor (Ras- GEF). Ras-GEF promotes the exchange of GDP and GTP. Once GTP bound, Ras is activated. 4. Ras-GTP initiates a further cascade of events that lead to changes in transcription and other effects. Note: SH2 domains specifically bind phosphotyrosine in the context of a surrounding sequence. 20  Canonical Map Kinase Pathway 21 MAP kinases The MAP-Kinase pathway MAP = Mitogen activated protein kinase 1. Raf is also known as MAP kinase kinase kinase. 2. A phosphorylation cascade (typically on ser/thr residues) is initiated resulting in phosphorylation and activation of MAP Kinase (ERK) 3. This in turn phosphorylates various target proteins and transcription factors. 4. For mitogen activation, these include genes required to progress into the cell cycle from G0 phase. 21  The Family of Small, monomeric G-proteins 22 The family of small G-proteins. 1. Small G-proteins require GEFs (guanosine exchange factors) to become activated. 2. Small G-proteins require a GAP to hydrolyze GTP to GDP and become inactivated. 3. Often GEFs and GAPs are strategically located to facility trafficking of vesicles or transport proteins, such as nuclear transport. Note: not all of the small G-proteins interact with RTKs; many of them act in other places and purposes. 22  Small G-proteins Require GAPs and GEFs Guanine nucleotide exchange factor (GEF) – Act like GPCRs do for trimeric G- proteins GTPase activating protein (GAP) GAPs and GEFs are specific for various small G-proteins 23 Mechanism of small G-proteins 1) Like large, trimeric G-proteins, they bind GDP and GTP, and are only active in the GTP-bound form. 2) The GDP-bound form is inactive. 3) Exchange of GTP for GDP is catalyzed by other proteins called GTP- exchange factors or GEFs. GEFs are typically specific for the small G-protein. Thus, in the previous slide, Sos is a Ras-GEF. It releases GDP and allows GTP to bind. 5) Ras-GAP will cause bound GTP to be hydrolyzed to GDP and terminate the signal. Important distinctions and comparisons: 1. Small g-proteins require guanine nucleotide exchange factors to switch GTP in and GDP out. They act as GPCRs do for the trimeric G-proteins but are typically small soluble or peripheral proteins regulated by other mechanisms inside the cell. 2. Unlike large, trimeric G-proteins, small G-proteins do not have a fully complete intrinsic GTPase. The GTPase activity requires complementary residues supplied by a GTPase activating protein (GAP). So small G-proteins 23 will stay GTP-bound, and active unless acted upon by a GTPase activating protein (GAP). 1. Note: there are known mutations in Ras that prevent it’s GTPase activity, and renders it constitutively active after activation (ie, GTP becomes bound and is not hydrolyzed even in the presence of a GAP.) Such mutations are commonly found in cancers. 23  PDGF Receptor 24 Platelet-derived Growth Factor and its Receptor PDGF activates phospholipase Cg and mobilizes calcium. Activates a GAP – turns off G-protein signaling Turns on a PI3 Kinase PI3-kinases – Phosphorylate the 3-position of Phosphatidyl Inositol phosphates. 24  PI3-Kinases Leads To Recruitment of Additional Proteins 25 PI3-kinase PI3 kinases phosphorylate PI molecules at the 3-position of inositol. 1. PI3Ks can be activated through G-protein signaling or through RTK signaling (previous slide). 2. PIP3 (marked in orange) and other inositides phosphorylated at the 3 position can recruit proteins to the plasma membrane through specific binding. 3. Proteins that bind PIP3 contain pleckstrin homology domains, a binding domain for PIP3. 4. Proteins can alter function or bring functionalities together via translocation to the membrane. 5. Some Proteins with pleckstrin homology domains are: 1. AKT (also called protein Kinase B); it binds PIP3 and also PI-3,4- bisphosphate. 2. PDK1 – phosphoinositide dependent protein kinase 1. PDK1 phosphorylates AKT among other proteins. 3. Bruton’s Tyrosine Kinase 4. General receptor for phosphoinositides (GRP1). 25 6. Pathways that include PI3-kinases are important in the following events: 1. Proliferation 2. Survival 3. Apoptisis (specifically ceramide induced apoptosis). 4. Mutations in kinases appear in some cancers – PI3 kinases contribute to cellular transformation and the development of cancer. 25  Survival By Prevention Of Apoptosis 26 PI3-Kinase pathway Example: Involvement of PI3-kinases in apoptosis. Steps: 1. RTK detects a ligand binding event 2. PI3-kinase is recruited by binding. 3. The activated PI3 kinase phosphorylates PIP2 and makes PIP3 4. PIP3 can then be bound and recruits AKT and PDK1 to the plasma membrane. 5. PDPK1 phosphorylates AKT and mTOR phosphorylates AKT 6. AKT phosphorylates Bad 7. Bad dissociates from apoptosis inhibitory protein and activates it. 8. The active apoptosis inhibitory protein prevents apoptosis. The PI3 Kinase/Akt/Tor pathway mediates the effects of many growth factor receptors. The insulin receptor works through this pathway to promote growth, and to signal up-regulation of glucose transporters. 26  Cross-talk among Signaling Pathways 27 Pathway Crosstalk Biochemical signaling pathways rarely work linearly, nor do they often work isolated and independent of other signaling inputs. Cell may receive many inputs simultaneously that impact the same pathways. Therefore, cells have to have mechanisms that integrate the signaling and decide the net output. Often, one pathway will predominate but others may have a synergistic effect (enhance it), or a moderating effect (inhibit it). Pathways can be very complex 1. Often pathways are presented as a linear series of events. 2. Many receptors and pathways have points of divergence that activate several separate branches. 3. The signaling paths are sometimes influenced, impacted, or nullified by input through other receptor mechanisms. 4. The purpose of such crosstalk is not always obvious, but there are a number of thoughts: 1. Some pathways can be activated through multiple mechanisms. Sometimes distinct responses in the same end-pathway can be achieved from distinct inputs. These can differ in intensity or temporal 27  nature. 2. Single inputs can activate multiple outputs. 3. Responses to one input can be modulated by another. 4. Redundancy to render the system more robust. 27  Summary Calcium is a major second messenger – Calcium signals a broad range of mechanisms including: Contraction, Vesicle fusion, Electrical signal propagation, Motility, Signal Integration – Calcium is stored in the ER/sarcoplasmic reticulum and in mitochondria – Calmodulin is a major mediator of calcium signaling. – IP3 is released from lipid (PIP2) and releases Ca2+ from internal stores (ER) – Diacyl Glycerol and Ca2+ activate Protein Kinase C NO – nitric oxide mediates smooth muscle relaxation. Prostaglandin synthesis is activated by arachidonic acid release from lipid by PLA2 RTK – Tyrosine Kinase receptors act via dimerization mechanisms – Activate tyrosine kinase activity. – Tyrosine phosphorylation promotes binding of other signaling proteins. – Activates many cascades – often changes protein expression – Involves intermediate signaling proteins such as the small G-proteins RAS and Raf. MAP Kinase cascade – Mitogen Activated Protein Kinase pathway 28 28