Cell Signaling and Signal Transduction (Chapter 15.2 PDF)

CHAPTER 15 Cell Signaling and Signal Transduction: Communication Between Cells Required reading: relevant sections of Chapter 15 15.2-1  15.1 | The Basic Elements of Cell Signaling Systems Extracellular signal to cellula...

CHAPTER 15 Cell Signaling and Signal Transduction: Communication Between Cells Required reading: relevant sections of Chapter 15 15.2-1  15.1 | The Basic Elements of Cell Signaling Systems Extracellular signal to cellular response – general principles 1: synthesis of the signaling molecule 2: release of the signaling molecule via exocytosis 3: transit of signaling molecule to the target cell 4: binding of signaling molecule (ligand) to a protein receptor on the target cell 5: binding of ligand to receptor results in a conformational change of the receptor 6: receptor initiates one or more intracellular pathways that results in changes in: cellular function Metabolism gene expression 7: deactivation of the receptor Shape movement 8: removal of ligand 15.2-2  15.1 | The Basic Elements of Cell Signaling Systems Molecular switches: phosphorylation The addition of phosphate groups to hydroxyl groups on (most commonly) serine threonine tyrosine Kinases phosphorylate Phosphatases dephosphorylate Phosphorylation changes a proteins charge and generally leads to a conformation change which can alter ligand binding or other features of the protein resulting in an increase OR decrease of its activity Phosphorylation is part of (almost) all signaling pathways Human genome contains 600 protein kinases 15.2-3 100 protein phosphatases  15.1 | The Basic Elements of Cell Signaling Systems Molecular switches: GTP-binding proteins GTPase superfamily: enzymes that hydrolyze GTP to GDP Two conformations on = bound GTP that modulates the activity of specific target proteins to which they bind off = bound GDP GAPS: GTPase-activating proteins RGSs: regulators of G protein signaling Remember that GDI: guanine nucleotide dissociation GDP is exchanged inhibitors for GTP GEFs: guanine nucleotide exchange factors 15.2-4  15.5 enzyme coupled receptors are transmembrane proteins that bind ligands cytosolic domain either contains an intrinsic kinase activity or associates directly with a kinase A kinase that associates with a receptor or is involved in a signal transduction pathway downstream is a non-receptor kinase The most common are the non - The most common are the Receptor Tyrosine Kinases Receptor Tyrosine Kinases (RTKs) (cytosolic) The human genome encodes nearly 60 RTKs and 32 non-receptor TKs 15.2-5 15.5 The most common are the Receptor Tyrosine Kinases (RTKs) Receptors bind ligands and the receptors are also tyrosine kinases Note the terminology of Receptor Tyrosine Kinases (RTKs): a tyrosine kinase receptor would be a receptor that had a tyrosine kinase as a ligand which is NOT what these receptors are – they themselves Feature: Function: have tyrosine kinase Extracellular domain (usually Binds ligand activity and are glycosylated) therefore Receptor Single transmembrane helix Participates in dimerization Tyrosine Kinases (RTKs) Dimerization/Aggregation activates the RTK (generally) Large cytosolic domain Location of the kinase and phosphorylation target sites 15.2-6 15.5 | Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Receptor Dimerization note that the ligand itself can be a dimer a monomeric ligand can interact with two receptor monomers two monomeric ligands can bind independently to to receptor monomers promoting dimerization 15.2-7 15.5 | Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Protein Kinase Activation Each monomer has both a tyrosine kinase activity AND individual tyrosines to be phosphorylated When the tyrosine kinase from one monomer phosphorylates the tyrosine residues on the other identical monomer = trans-autophosphorylation Note that these phosphorylated tyrosine can now act as docking sites for other proteins 15.2-8 15.5 | Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Phosphotyrosine-Dependent Protein-Protein Interactions Phosphorylated tyrosines bind effector proteins that have either a Src-homology 2 (SH2) domain a phosphotyrosine-binding (PTB) domain SH2 domains are composed of roughly 100 amino acids and contain a conserved binding-pocket that accommodates a phosphorylated tyrosine residue Protein with an SH2 domain (purple) binding a phosphotyrosine containing PTB (phosphotyrosine binding) peptide – the phosphorylated tyrosine domains can bind to phosphorylated (blue) is key to binding tyrosine residues and are usually present as part of an Asn-Pro-X-Tyr motif 15.2-9 common domains and what they bind to The now phosphorylated tyrosines become binding sites for other molecules Each molecule that binds must recognize the phosphorylated tyrosine in the context of other specific amino acids 15.2-10 15.5 | Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Activation of Downstream Signaling Pathways SH2 and PTB domain proteins are: 1) Adaptor proteins that bind other proteins 2) Docking proteins that supply receptors with other tyrosine phosphorylation sites 3) Signaling enzymes (kinases) that lead to changes in cell 4) Transcription factors A diversity of signaling proteins Cells contain numerous proteins with SH2 or PTB domains that bind to phosphorylated tyrosine residues 15.2-12 15.5 | Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Ending the Response Signal transduction by RTKs is usually terminated by internalization of the recepto primarily through clathrin-mediated endocytosis Some RTKs may bind to the clathrin adaptor protein AP-2, or may be targeted by ubiquitination by ubiquitin ligases through SH2 domains or adaptor proteins Internalized RTKs can have several alternate fates; degraded in lysosomes returned to the plasma membrane the Ras-MAP kinase pathway is probably the best characterized signaling cascade What is SRC? it’s a tyrosine kinase Is it an RTK? no - it’s a non-receptor tyrosine kinase it’s involved in signal transduction pathways SRC has 4 domains: 2 of these domains are involved in binding to other proteins these two domains turn out to be very common (conserved) in proteins involved in binding to other proteins in signal transduction These domains were named SH2: src-homology 2 SH3: src-homology 3 so based on nomenclature, even 15.2-13 SRC has SH2 and SH3 domains Cancer-critical genes (genes whose alteration frequently contributes to causing cancer) Gain-of-function mutation: proto-oncogenes that mutate into oncogenes Mutation of a single copy of a proto- oncogene that converts it to an oncogene has a dominant, growth- promoting effect on a cell terminology: when a normal gene mutates into a gain-of-function gene, that gene is now called an oncogene The normal gene is called a proto-oncogene same applies to the protein products: oncogenes make oncoproteins proto-oncogenes (normal genes) make proto-oncoproteins (normal proteins) 15.2-14 Discovery of oncogenes Viruses have no role in the majority of human cancers BUT they are prominent causes of cancer in some animal species Through study of these animal tumor viruses that oncogenes were discovered Chickens are subject to infections that cause connective-tissue tumors (sarcomas) The infectious agent was characterized (1911) by Peyton Rous and called the Rous Sarcoma Virus (RSV) The question then became “what part of the viral genome caused this cancer in chickens?” 15.2-15  Genomes of vertebrate cells contain sequences similar to (but not identical to) v-src Rous Sarcoma Virus would have initially picked up the normal cellular gene counterpart to v-src which is called c-src (cellular src) c-src would be considered the proto- oncogene that then underwent mutations to become the oncogene (v-src) c-Src is a non-receptor tyrosine kinase (remember - that means it’s cytosolic and not a transmembrane receptor) Phosphorylation of c-Src at Tyr527 inactivates it’s tyrosine kinase activity More oncogenes have since been discovered 15.2-16 Discovery of oncogenes RSV (an RNA virus) is a retrovirus whose RNA genome is reverse transcribed into DNA and inserted into the host genome where it can persist and be inherited by subsequent generations of cells The cancer causing DNA turned out to be DNA that was not even part of the required viral DNA - it was a piece of DNA that was a “passenger” - something the virus had picked up at some point -discovered and sequenced in the 1980’s – it had an extra gene named v-src (viral sarcoma) 15.2-17 Following the discovery of v-src (the oncogene) and c-src (the proto-oncogene) many other viral oncogenes were identified The question then became - how did all this relate to human cancers since human cancers are not infectious and retroviruses play no role To answer this question, researchers began directly searching for oncogenes in the genomes of human cancer cells How? by looking for DNA fragments from cancer cells that would induce uncontrolled cell growth in non-cancer cells 3T3 cells = mouse fibroblasts chosen because they proliferated indefinitely in (tissue) culture - meaning that they were somehow already altered and thought to be more susceptible to the addition of oncogenic DNA 15.2-18 DNA from cancer cells is fragmented and introduced into mouse 3T3 fibroblasts result: occasional colonies of abnormally proliferating cells began to appear This (human) DNA fragment could then be isolated from the mouse cells These initial experiments demonstrated that each colony was a clone originating from a single cell that had incorporated a DNA fragment that drove this cancerous behaviour And once this DNA fragment was isolated and sequenced it turned out to be a human version of a gene already known What was this proto-oncogene? 15.2-19 more oncogenes were isolated remember - these are all gain of function mutations as more oncogenes were isolated more sophisticated analysis began to dissect the different ways that proto-oncogenes can turn into oncogenes 15.2-20  15.5 | Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction Protein Kinase Activation Once trans-autophosphorylation of the receptors occurs the receptor recruits cytosolic proteins The cytosolic proteins have amino acid stretches (domains) that recognize the phosphotyrosine and nearby residues on the receptor One such domain is called the SH2 domain because the residues are similar to sequences on Src (SH = Src Homology) Multiple pathways can be activated! This is an example of cross-talk between signaling pathways Almost all RTKs can activate the Ras/MAP kinase pathway 15.2-21  15.5 | The Ras-MAP Kinase Pathway Ras was originally described as the product of a retroviral oncogene and later determined to be derived from its mammalian host Approximately 30 percent of all human cancers contain mutant versions of RAS genes Ras proteins are part of a superfamily of at least 167, monomeric G proteins (GTPases) involved in the regulation of numerous processes, including cell division, differentiation, gene expression, cytoskeletal organization, vesicle trafficking, and nucleocytoplasmic transport Ras is a small GTPase that is anchored at the inner surface of the plasma membrane by a covalently attached lipid group that is embedded in the inner leaflet of the bilayer The GTPase Ras mediates signaling by most RTKs 15.2-22 15.2-23 There are three members of the RAS family that are altered in up to 30% of all cancers 15.2-24 Ras activates a MAP kinase signaling cascade This signal transduction system is highly conserved from yeast to humans Ras recruits Raf to the plasma membrane and helps activate Raf Raf then activates Mek Mek activates Map kinase (Erk) Map kinase then phosphorylates a variety of downstream proteins including other protein kinases as well as gene regulatory proteins in the nucleus How does Ras activate Raf? 15.2-25  Oncoprotein derived from an oncogene Mutated Ras protein hydrolyzes bound GTP very slowly = constituitively active Ras 15.2-26 Mutations in any part of the Ras-Map pathway can be oncogenic 15.2-27 pathway specific targets 15.2-28 Drugging the undruggable RAS: Mission Possible? Adrienne D. Cox, Stephen W. Fesik, Alec C. Kimmelman, Ji Luo & Channing J. Der Nature Reviews Drug Discovery 13, 828–851 (2014) doi:10.1038/nrd4389 Published online 17 October 2014 Despite more than three decades of intensive effort, no effective pharmacological inhibitors of the RAS oncoproteins have reached the clinic, prompting the widely held perception that RAS proteins are 'undruggable'. However, recent data from the laboratory and the clinic have renewed our hope for the development of RAS- inhibitory molecules. In this Review, we summarize the progress and the promise of five key approaches. Firstly, we focus on the prospects of using direct inhibitors of RAS. Secondly, we address the issue of whether blocking RAS membrane association is a viable approach. Thirdly, we assess the status of targeting RAS downstream effector signalling, which is arguably the most favourable current approach. Fourthly, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, RAS-mediated changes in cell metabolism have recently been described and we discuss whether these changes could be exploited for new therapeutic directions. We conclude with perspectives on how additional complexities, which are not yet fully understood, may affect each of these approaches 15.2-29 Nature: 2019 Nov;575(7781):217-223. The Clinical KRAS(G12C) Inhibitor AMG 510 Drives Anti- Tumour Immunity Canon J1, Rex K2, Saiki AY2, Mohr C2, Cooke K2, Bagal D3, Gaida K2, Holt T2, Knutson CG4, Koppada N4, Lanman BA2, Werner J2, Rapaport AS3, San Miguel T2, Ortiz R4,5, Osgood T2, Sun JR2, Zhu X4,6, McCarter JD2, Volak LP4,7, Houk BE8, Fakih MG9, O'Neil BH10, Price TJ11,12, Falchook GS13, Desai J14, Kuo J15, Govindan R16, Hong DS17, Ouyang W3, Henary H8, Arvedson T3, Cee VJ2, Lipford JR18 Abstract KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking. 15.2-30 On December 12, 2022, the Food and Drug Administration (FDA) granted accelerated approval to adagrasib (Krazati, Mirati Therapeutics, Inc.), a RAS GTPase family inhibitor, for adult patients with KRAS G12C¬-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC), as determined by an FDA-approved test,... 15.2-31  15.5 | The Ras-MAP Kinase Pathway Adapting the MAP Kinase to Transmit Different Types of Information Scaffolding proteins function to tether members of a signaling pathway in a specific spatial orientation to enhance their mutual interactions (e.g. AKAPs) Some scaffolds can induce a conformation change in signaling proteins, leading to activation or inhibition Some scaffolds have enzymatic activity (e.g. yeast Pbs2) Scaffolds can prevent proteins from participating in other pathways, resulting in higher specificity 15.2-32 Rho family GTPases functionally couple cell surface receptors to the cytoskeleton Rho family includes: Rho, Rac, CDC42 regulate actin and microtubules: controlling shape, motility, adhesion help regulate: cell-cycle progression gene transcription membrane transport AND MORE While Ras is always membrane associated inactive Rho family GTPases often are bound to a guanine nucleotide dissociation inhibitor (GDI) in the cytosol The GDI prevents Rho from interacting with it’s GEF at the plasma membrane 15.2-33 15.8 Convergence, Divergence, and Cross-Talk among Different Signaling Pathways GPCRs and RTKs activate overlapping signaling pathways 15.2-34 15.2-35 Learning objectives: Understand what an enzyme coupled receptor is Understand what a receptor tyrosine kinase is Understand what a non-receptor kinase is Understand the characteristics of RTKs Understand what SRC is Understand the role of src homology domains and PTB domains Know the steps of the Ras-Map pathway beginning with ligand binding Know the general role of Rho and what prevents it from binding to the membrane when inactive Other terms to be familiar with: Oncogene Proto-oncogene Onco-protein Proto-oncoprotein Gain of function mutation 15.2-36