Cell Signaling - Signal Transduction Notes PDF

Burman University, BIOL 373 Cellular Biology, Page 1 Cell Signaling – Signal Transduction (Topic 10) Learning Objectives: 1. Summarize the steps in a typical signal transduction pathway, beginning with a signal originating outside the cell to the intracellular signaling events th...

Burman University, BIOL 373 Cellular Biology, Page 1 Cell Signaling – Signal Transduction (Topic 10) Learning Objectives: 1. Summarize the steps in a typical signal transduction pathway, beginning with a signal originating outside the cell to the intracellular signaling events that lead to changes in cellular metabolism, function and movement, or modification of gene expression and development. 2. Describe the properties of receptor-ligand interactions. 3. Provide examples of each of the following major classes of receptors and compare and contrast their mechanism of signaling and termination: G-Protein coupled receptors, receptor tyrosine kinases, receptors that lead to ubiquitination of signaling proteins, and receptors that binding to signals attached to adjacent cells. 4. Define the terms used in signal transduction – for example, GPCR, G-Protein, second messenger, protein kinase/phosphatase, adapter proteins, effector proteins, signal amplification, Hedgehog, JAK/STAT, MAP kinase, RTK, Ras protein, SH domains, Wnt, NF-kB, Notch/Delta and more. 5. Describe the role of these signal transduction pathways in controlling normal cellular responses and gene expression. 6. Describe how alterations in some signal transduction pathways that can lead to human disease. Text: Lodish et al. (2021), Ch. 15 (S15.1-4) & 16 (S16.1-2, 16.6 and 16.7) Verse: The words of the reckless pierce like swords, but the tongue of the wise brings healing. Proverbs 10:19 Like communication between and within cells, proper human communication is important especially in this increasing turbulent world. Our words should be truthful, accurate and sensitive to all peoples – something that is lacking in our world. Let us speak healing words. Introduction: Cell Signaling & Signal Transduction § Signal transduction is the process of converting extracellular signals into cellular responses. § Extracellular signaling molecules regulate interaction between unicellular organisms and are critical regulators of physiology and development in multicellular organisms. § No cell lives in isolation! § Topic Outline I. Signal Transduction: From Extracellular Signal to Cellular Response (15.1 & 15.2) II. G Protein-coupled Signal Transduction Pathway (15.3 & 15.4) III. Receptor Tyrosine Kinase Signal Transduction Pathway (16.1 & 16.2) IV. Notch/Delta Signaling Pathway (16.6) V. Signaling controlled by Ubiquitination (16.7) LW2024 Burman University, BIOL 373 Cellular Biology, Page 2 I. Signal Transduction [S15.1 & 15.2] A. Depending on the nature of the signal, communication by extracellular signals usually involves common steps (F15-3). § Hydrophobic signaling molecules, such as steroids, thyroxine, and retinoic acid, etc., can diffuse across the plasma membrane (1; numbers correspond to numbers in F15-3) and bind to cytoplasmic receptors (2). The receptor-signal complex moves into the nucleus (3), where it can bind to transcription control regions in DNA and activate/repress gene expression (left side, F15-3). § Some signals bind to cell-surface receptors (right side, F15-3): 1. Hydrophilic molecules: – Membrane-anchored and secreted proteins – Peptides (e.g., insulin, glucagon, growth factor) – Small hydrophilic (charged) molecules derived from amino acids (e.g., catecholamines) 2. Lipophilic molecules that bind to cell-surface receptors (e.g., prostaglandins) § The majority of signaling molecules cannot diffuse across the membrane, binding to specific cell-surface receptors, trigger a conformation change in the receptor, thus activating it (4). § The activated receptor then initiates an intracellular signal transduction pathway(s) by activating one or more signal transducing proteins and/or 2° messengers (5). § Eventually leads to activation of one or more effector proteins (6). § A short-term change in cellular metabolism, function, or movement (7a); or, a long-term change in gene expression or development (7b); § Termination or down-modulation of the cellular response caused by negative feedback from intracellular molecules (8); and removal of the extracellular signal, which usually terminates the cellular response (9). B. Signaling molecules operate over various distances in animals (F15-2): § Signaling by membrane-attached signal on adjacent cells are also called juxtaposed signaling. LW2024 Burman University, BIOL 373 Cellular Biology, Page 3 C. Receptor proteins § Located on cell surfaces of target cells or intracellularly in the cytosol or nucleus. § In most cases, the ligand’s only function is to bind the receptor, changing the receptor properties upon binding. § Bind to one or a few related ligands. § Exhibit ligand-binding specificity and effector specificity. § Ligand specificity is determined by noncovalent interactions between the ligand and specific amino acids in the receptor protein (molecular complementarity; F15-4). § The maximal response of a cell to a particular ligand generally occurs at [ligand]s at which most of its receptors are still not occupied (F15-8). § The [ligand] at which half its receptors are occupied, Kd, is a measure of the affinity of the receptor for the ligand. § Receptors activate a limited number of signaling pathways resulting in effector specificity. § The target cells often modify or degrade the ligand and/or receptor, and in doing so can modify or terminate their response. § Major classes of cell-surface receptors and their pathways (F16-1). D. Intracellular Signal Transduction include the following features: § 2nd messengers and signal amplification. § Conserved intracellular proteins including GTPase switch proteins, kinases, phosphatases and adapter proteins/domains. § Resetting or termination of the signal. § Regulation and interaction of multiple signaling pathways. § The effects of many hormones are mediated by second messengers [F15- 5]. – 2nd messengers are intracellular signaling molecules that carry signals from the receptors. LW2024 Burman University, BIOL 373 Cellular Biology, Page 4 – Concentrations of 2nd messengers are modulated in response to binding of ligand (i.e., the 1st messenger) to cell-surface receptors. – 2nd messengers regulate activities of enzymes and nonenzymatic proteins called effectors. – Signaling pathways involving 2nd messengers and enzyme cascades can amplify an external signal tremendously [F15-23]. § GTPase switch proteins – GTPase switch proteins are conserved GTP-binding proteins that act as molecular switches. – “On” when bound to GTP and “off” when bound to GDP [F15-7]. – Signal-induced conversion of the inactive to active state is mediated by a guanine nucleotide-exchange factor (GEF). – These proteins possess intrinsic GTPase activity. – The rate of GTP hydrolysis is usually enhanced by a GTPase-accelerating protein (GAP). – e.g., trimeric G proteins [S15.3] and monomeric Ras or Ras-like proteins [S16.3]. § Protein kinases – Could be part of the receptor itself or found in the cytosol or associated with the plasma membrane. – Most phosphorylate Ser/Thr and Tyr. § Protein phosphatases – Phosphatases remove phosphate groups. – Can act together with kinases to switch proteins on or off. – Some proteins are activated when phosphorylated and inactivated when dephosphorylated; some proteins have the opposite pattern. – Kinase and phosphatase activities are usually highly regulated. § F15-7 (old text). Signal transduction pathway involving a G protein, 2° messenger, a protein kinase and target proteins. LW2024 Burman University, BIOL 373 Cellular Biology, Page 5 § Adapter proteins – No catalytic activity. – Coordinate the formation (via docking sites) of multicomponent signaling complexes. – The clustering of membrane proteins, to enhance signal transduction and to bring several signaling components/molecules close to the membrane, can occur with proteins that have adapter domains. – These adapter domains can organize the proteins in the signaling complex. – e.g., PDZ domain in cytosolic proteins bind to C-terminals of integral membrane proteins. E. Regulation of Pathways § The ability of cells to respond appropriately depends on the regulation of the signaling pathways. § Once a particular ligand is withdrawn, rapid termination occurs (via degradation of 2nd messenger, deactivation of a signaling protein, etc). § Receptor desensitization at high [ligand]s or after prolonged exposure is also important. § Common signaling ends are initiated by different receptors, or many signaling pathways can be initiated from one receptor class. § Extracellular signals are often integrated into complex regulatory networks [F16-41, old text]. II. G Protein-coupled Signal Transduction Pathway (S15.3 & 15.4) § Trimeric G proteins transduce signals from coupled cell-surface receptors to associated effector proteins, which are either enzymes that form second messengers (e.g., cAMP and Ca2+), or cation channel proteins (e.g., acetylcholine receptor). § Features of GPCR signaling pathways 1. A receptor that contains seven membrane-spanning domains. – G protein-coupled receptor (GPCR; F15-12; T15-1). These are further divided into 3 classes according to how they bind to their ligands (F15-13). LW2024 Burman University, BIOL 373 Cellular Biology, Page 6 2. A coupled trimeric G protein, which functions as a switch by cycling between active and inactive forms. – Different G proteins are activated by different GPCRs and in turn regulate different Effector proteins (T15-2). – Signals most commonly are transduced by Ga, a GTPase switch protein that alternates between an active (“on”) state with bound GTP and inactive (“off”) state with GDP. – The b and g subunits of the G protein, which remain bound together, occasionally transduce signals. – Hormone-occupied receptors act as GEFs for Ga proteins, catalyzing the dissociation of GDP and enabling GTP to bind. – The resulting change in conformation of switch regions in Ga causes it to dissociate from the Gbg subunit and interact with an effector protein (F15-16a; F15-14, old text). LW2024 Burman University, BIOL 373 Cellular Biology, Page 7 3. A membrane-bound effector protein. – Adenylyl Cyclase is activated by GPCRs (F15-26, old text) – Adenylyl cyclase is stimulated and inhibited by different receptor-ligand complexes (F15-20). 4. Feedback regulation and desensitization of the signaling pathway (see below). 5. A second messenger is present in many GPCR pathways. In many cases it is cAMP (F15-18). – Gsa, which is activated by multiple types of GPCRs, binds to and activates adenylyl cyclase, enhancing the synthesis of 3’,5’-cyclic AMP, a 2nd messenger. – cAMP phosphodiesterase hydrolyzes cAMP to AMP thus terminating the activity of the 2nd messenger. – cAMP-dependent activation of protein kinase A (PKA; F15-21a) mediates the diverse effects of cAMP in different cells. cAMP binding releases the catalytic subunits. – The substrates for PKA and thus the cellular response to hormone-induced activation of PKA vary among cell types (T15-3). – In liver and muscle cells, activation of PKA induced by epinephrine (e.g., released when running away from a bear) and other hormones exerts a dual effect, inhibiting glycogen synthesis and stimulating glycogen breakdown via a kinase cascade (F15-19, F15- 22). – PKA promotes glycogen degradation PKA phosphorylates and thus activates an intermediate kinase, glycogen LW2024 Burman University, BIOL 373 Cellular Biology, Page 8 phosphorylase kinase (GPK), that in turn phosphorylates and activates glycogen phosphorylase (GP), the enzyme that degrades glycogen. PKA also phosphorylates and inactivates glycogen synthase (GS), the enzyme that synthesizes glycogen, and the inhibitor protein (IP) of phosphoprotein phosphatase (PP). – Phosphoprotein phosphatase (PP) stimulates glycogen synthesis In the absence of cAMP (inactive PKA), PP is active. PP removes the phosphates from inactive GS, thereby activating it, and from the active forms of GPK and GP, thereby inactivating them. Such coordinate regulation of synthetic and degradative pathways provides an efficient mechanism for a particular cellular response. § Integration of Multiple 2˚ Messengers Regulates Glycogenolysis. – To respond to a complex environment a cell senses and integrates its responses to more than one signal. – These responses may be different in different tissues. – For example, in the liver cell, hormonal stimulation also results in the secretion of Ca2+ ions that activates GPK and inhibits protein kinase C that can inhibit GS (F15- 32). § Mechanisms to Terminate Signaling from GPCRs 1. Affinity of the receptor for its ligand decreases when the GDP on Gas is replaced with GTP - resulting in the dissociation of the ligand from the receptor. 2. Intrinsic GTPase activity of Gas converts GTP to GDP, resulting in the inactivation of the Gas protein and hence decreased adenylate cyclase activity. Adenylate cyclase also functions as a GAP for Gas. 3. cAMP phosphodiesterase hydrolyzes cAMP to 5’-AMP, terminating the cellular response. – Therefore, continual presence of hormone is required for continued activation of adenylate cyclase and elevated cAMP levels. – cAMP phosphodiesterase is often colocalized together with PKA. – Localization of cAMP to specific regions of the cell by anchoring proteins [F15-25]. mAKAP is an A kinase- associated protein that anchors both PKA and cAMP phosphodiesterase. LW2024 Burman University, BIOL 373 Cellular Biology, Page 9 Anchoring proteins restricts the effects of cAMP to particular subcellular locations. 4. Receptor desensitization (F15-26) – BARK (b-adrenergic receptor kinase) phosphorylates the activated GPCR receptors, leading to the binding of b-arrestin and endocytosis of the receptors. – b-arrestin is a cytosolic protein that binds to receptors extensively phosphorylated by BARK and completely inhibits their interaction with and ability to activate Gs. – The consequent reduction in cell-surface-receptor numbers renders the cell less sensitive to additional hormone. § Activation of gene transcription by G protein- coupled receptors (F15-24). – Signal-induced activation of PKA often leads to phosphorylation of CREB (CRE binding protein). – CREB links cAMP and PKA to activation of gene transcription. – Phosphorylated CREB associates with coactivator CBP/P300 to stimulate the transcription of target genes controlled by the CRE regulatory element. § GPCR-Bound Arrestin Activation of Other Kinase Cascades – The GPCR-arrestin complex also acts as a scaffold for binding and activating several other cytosolic kinases (F15-26). – These include c-Src, a protein kinase that activates the MAP kinase pathway leading to transcription of genes needed for cell division. – Another cascade result in the activation of c-Jun, a transcription factor that promotes the expression of certain growth-promoting enzymes and stress proteins. III. Receptor tyrosine kinase pathways (16.1 and 16.2) § Receptor tyrosine kinases recognize soluble, or membrane bound peptide/protein hormones, that act as growth factors (F16-1a). § Binding of the ligand stimulates the receptor’s tyrosine kinase activity, which leads to the activation of signaling cascades that result in gene expression. § RTK pathways are involved in regulation of cell proliferation and differentiation, promotion of cell survival, and modulation of cellular metabolism. LW2024 Burman University, BIOL 373 Cellular Biology, Page 10 § Induction of genes by pathways that activate transcription factors also depends on the presence of binding sites for the factors, the epigenetic state, and the presence of the general transcription factors and or nuclear proteins (F16- 2). § RTKs directly phosphorylate and activate signaling proteins, including transcription factors located in the cytosol (F16-1a; eg., STAT proteins, F16-20a). § Some RTKs transmit a hormone signal to Ras, a small GTPase switch protein (monomeric G protein unlike the trimeric G proteins of GPCR; F16-12) that passes on the signal on to downstream components (F16-13; eg, Ras/MAP kinase pathway). § General structure and activation of RTKs [F16-3]. § Ligand binding leads to autophosphorylation of RTKs – Monomeric RTKs dimerize during binding to ligands. – Leads to activation of the intrinsic protein tyrosine kinase activity of the receptor and phosphorylation of Tyr residues in its cytoplasmic domain. – The activated receptor can also phosphorylate other protein substrates. § An adapter protein (GRB2) and GEF (Sos) link most activated RTKs to Ras [F16-10]. – Adapter protein has a SH2 and two SH3 docking sites. – The SH2 domain in GRB2 binds to a phosphotyrosine in activated RTKs, while its two SH3 domains binds Sos, bringing Sos close to the membrane-bound Ras- GDP (step 2). § Ras activation (step 3) – Sos binding Ras causes a large conformational change that permits release of GDP and binding of GTP, forming active Ras (i.e., Sos is a GEF). – Activated Ras induces a kinase signal cascade that LW2024 Burman University, BIOL 373 Cellular Biology, Page 11 culminates in activation of MAP kinase. § MAP kinase is a serine/threonine kinase that can translocate into the nucleus and phosphorylate many different proteins, including transcription factors that regulate gene expression. – Signals pass from activated Ras to Raf, MEK, and MAP kinase are sequentially phosphorylated [F16-13]. § Map kinase activation – Phosphorylation activates MAP kinases and many other protein kinases (e.g., MEK) involved in signal-transduction pathways. – Phosphorylation of MAP kinase enhances its catalytic activity and promotes dimerization. – Dimeric MAP kinase translocates into the nucleus. – MAP kinase regulates the activity of many transcription factors [TCF; F16-14]. – Activated MAP kinase can also phosphorylate p90RSK (another kinase), activating it. Phosphorylated p90RSK also migrates to the nucleus to phosphorylate SRF transcription factors. – Activated TCF, together with SRFs activate gene transcription. § Multiple MAP kinase pathways are found in eukaryotic cells – Scaffold proteins (e.g., Ste5 and Pbs2; F16-15) isolate multiple MAP kinase pathways by assembling large pathway- specific signaling complexes. – This assures that activation of one pathway by a particular extracellular signal does not lead to activation of other pathways containing shared components. LW2024 Burman University, BIOL 373 Cellular Biology, Page 12 IV. Notch/Delta Signaling Pathway (16.6) § Extracellular signaling molecule is on the membrane of a juxtaposed cell. § Upon binding to its ligand Delta on the surface of an adjacent cell, the Notch receptor protein undergoes two proteolytic cleavages [F16-25]. 1. Initially, the Notch receptor is folded and cannot be cleaved by ADAM 10, a matrix metalloprotease in the membrane. 2. Notch-Delta binding exposes the ADAM 10-cleavage site on Notch. 3. The released Notch extracellular domain remains bound to Delta and is endocytosed by the signaling cell. 4. Endocytosed Notch fragment and Delta are degraded in lysosomes. 5. Next the Nicastrin subunit, of the g-secretase complex, binds to the stump generated by ADAM 10; Presenilin 1, the protease subunit, cleaves Notch at the membrane. 6. The released Notch cytoplasmic segment, a transcription factor, then translocate into the nucleus and modulates gene transcription. V. Signaling controlled by Ubiquitination (16.7) § Many signaling pathways involve ubiquitination and proteolysis of target protein and so are irreversible or slowly reversible, e.g., Wnt and Hedgehog pathways. § Target proteins can be either a transcription factor or an inhibitor of a transcription factor. § Wnt controls numerous critical developmental events, like brain development, limb patterning and organogenesis. § Hedgehog functions as a morphogen during development. § Activating mutations in both pathways can cause cancer. § Both Hedgehog and Wnt are secreted proteins that have lipid anchors that tether them to cell membranes, thereby reducing their signaling ranges. § They bind to receptors that are like the 7-spanning G protein-coupled receptors, but do not activate G proteins. § In the resting state, key transcription factors in both pathways are ubiquitinated and targeted for proteolytic cleavage rendering them inactive. LW2024 Burman University, BIOL 373 Cellular Biology, Page 13 § Activation of the pathways involves dissociation of large cytosolic protein complexes, inhibition of ubiquitination, and release of the active transcription factor. § Kinases including glycogen synthase kinase 3 (GSK3) play important roles. A. Wnt Pathway § Wnt signals act through a cell-surface receptor Frizzled (Fz) and co-receptor LRP, and an intracellular complex containing b-catenin. § In the absence of Wnt, the transcription factor TCF is bound to promoters or enhancers of target genes, but is associated with transcription repressors such as Groucho (Gro) inhibiting transcription (F16-26a). – b-catenin is associated with Axin (a scaffolding protein), which forms a complex with APC, and kinases CK1 and GSK3, which sequentially phosphorylates b-catenin. – A ubiquitin ligase (E3 TrCP) binds to phosphorylated b- catenin, leading to ubiquitination and degradation in the proteasomes. § + Wnt – Wnt binding to Fz and LRP triggers phosphorylation of LRP by GSK3, allowing subsequent binding of Axin. – This disrupts the Axin-APC-CK1-GSK3-b-catenin complex, preventing the phosphorylation of b-catenin (and leads to its eventual accumulation). – After translocating into the nucleus, b-catenin binds to TCF to displace Gro repressor and recruiting other proteins to activate gene transcription (F16-26b). – Thus, Wnt signaling promotes the stabilization of a transcription factor (b-catenin) and frees it from a cytoplasmic complex to localize to the nucleus. B. Hedgehog Pathway in Drosophila § The Hedgehog signal also acts through 2 cell-surface proteins, Smoothened (Smo) and Patched (Ptc), and an intracellular complex containing Cubitis interruptus (Ci) transcription factor. § An activating form of Ci is generated in the presence of Hedgehog; a repressing Ci fragment (formed with ubiquitination and proteolysis) is generated in the absence of Hedgehog. § Unlike Wnt, Hedgehog (Hh), the signaling molecule, undergoes post-translational processing before it is active. § Hedgehog is formed from a precursor protein with autoproteolytic activity that enables the protein to be cleaved in half (F16-28). LW2024 Burman University, BIOL 373 Cellular Biology, Page 14 § The N-terminal fragment is covalently modified at the carboxy- and N-terminus with cholesterol and palmitoyl, respectively. – Modifications are performed by the C-terminal portion of the precursor. – The N-terminal, made more hydrophobic, tethers the secreted Hh protein to the plasma membrane, keeping its range of action to nearby. § Both Patched and Smoothened change their subcellular location in response to Hedgehog binding to Patched. § -Hh (F16-29a) – In the absence of Hh, Ptc inhibits Smo, which is present largely in the membrane of internal vesicles. – A complex of Fused (Fu; a kinase) with other kinases, Costal-2 (Cos2; a kinesin-related motor protein), and Ci (a zinc-finger transcription factor) binds to microtubules. – Ci is phosphorylated in a series of steps involving PKA, GSK3 and CK1 (Casein kinase I). – The phosphorylated Ci is proteolytically cleaved by the ubiquitin-proteasome pathway, generating Ci75, a transcriptional repressor of Hh-responsive genes. – Another regulator SUFU (Suppressor of Fused Homolog) may also associate with the full-length Ci to prevent nuclear translocation. § +Hh (F16-29b) – Hh binds to Ptc, causing some Ptc to move to internal compartments and relieving the inhibition of Smo. – Smo moves to the plasma membrane, is phosphorylated, binds Cos2, and is stabilized from degradation. Both Fu and Cos2 become extensively phosphorylated. – Fu-Cos2-Ci complex become stabilized, can become modified Ci*, which displaces Ci75 from the promoters, recruits CREB-binding activator protein (CRP), and induces expression of target genes. § Hedgehog Pathway in Vertebrates – Hh signaling in vertebrates requires primary cilia and intraflagellar transport proteins. Ptc localizes to the ciliary membrane in the absence of Hh and Smo moves to cilia when Hh is present. – Otherwise, the overall signaling pathway is like that in flies. – The transcription factor Gli is the vertebrate homolog of Ci. LW2024 Burman University, BIOL 373 Cellular Biology, Page 15 C. NF-kB Pathway § The NF-kB transcription factor regulates many genes that permit cells to respond to infection and inflammation. § In the NF-kB Pathway, the degradation of an inhibitor protein, I-kBa, activates the NF- kB transcription factor (F16-30). § The NF-kB transcription factor, composed of p50 and p65 subunits, are sequestered in the cytosol, bound to I-kBa. § NF-kB Pathway Steps: 1. Activation of the trimeric I-kB kinase (IKKa, IKKb and NEMO) is stimulated by many agents, including virus infection, ionizing radiation, binding of pro- inflammatory cytokines TNFa or IL-1 to their respective receptors, or the activation of several Toll-like receptors by components of the invading bacteria or fungi. 2. The b subunit of I-kB kinase (IKKb) phosphorylates the inhibitor I-kB, which binds to E3 Ubiquitin ligase. 3. Polyubiquitination of I-kBa 4. targets I-kBa for degradation by proteasomes. 5. I-kBa removal unmasks the nuclear-localization signals (NLS) in both p50 and p65, allowing their translocation into the nucleus. 6. In the nucleus, NF-kB activates the transcription of many genes (genes of inflammatory cytokines, etc.), including the gene of I-kBa, which acts to terminate signaling. § The activation of receptors, catalyze the formation of polyubiquitin chains that serve as scaffolds linking receptors to downstream proteins (e.g., I-kB kinase) in the NF-kB pathway (F16-31). § In summary: We only touched on a few signal tranducing pathways. LW2024