Cell Communication and Signaling Pathways PDF

Cell communication and signaling Pathways in normal cells S2P3U3 Instructor: Ms. Simone Vaz 1  Cellular Communication Everything an animal does involves communication among cells e.g., moving, digesting food...

Cell communication and signaling Pathways in normal cells S2P3U3 Instructor: Ms. Simone Vaz 1  Cellular Communication Everything an animal does involves communication among cells e.g., moving, digesting food Cell signaling – communication between cells Signaling cell: sends a signal (usually chemical) Target cell: receives the signal 2 3 4  What is the Signal? External message to the cell Peptides / Proteins- Growth Factors Amino acid derivatives - epinephrine, histamine Other small biomolecules - ATP Steroids, prostaglandins Gases - Nitric Oxide (NO) Photons Damaged DNA Odorants, tastants Signal = LIGAND Ligand- A molecule that binds to a specific site on another molecule, usually a protein, ie receptor 5  Types of Cell Signaling Direct: via gap junctions Indirect: signaling cell releases a chemical messenger that binds to a receptor on the target cell and activates a signal transduction pathway Short distances Intracrine: Produces and effects the same cell internally Juxtacrine: ligand on one cell surface binds to a receptor on the other adjacent cell. Paracrine: diffusion to a nearby cell (Neural: electrical signal travels along a neuron and releases a neurotransmitter) Autocrine: diffusion back to the signaling cell via receptors Long distances Endocrine: hormone is transported by the circulatory system 6 Direct Signaling 7 Direct Signaling GAP JUNCTIONS Gap junctions – specialized protein complexes that create an aqueous pore between two adjacent cells Hydrophilic chemical messengers can travel through the lipid membrane Typically involves the movement of ions 8  Direct Signaling GAP JUNCTIONS Gap junctions allow signaling information to be shared by neighboring cells Ca2+, cAMP etc. but not for proteins or nucleic acids Connexin 43 deficiency --- Various disorders including abnormal heart development “Bystander effect” mediated by gap junctions 9 Indirect Signaling 10  Autocrine and Paracrine Because autocrine signaling is most effective when carried out simultaneously by neighboring cells of the same type, it may be used to encourage groups of identical cells to make the same developmental decisions. Thus autocrine signaling is thought to be one possible mechanism underlying the "community effect" observed in early development, where a group of identical cells can respond to a differentiation-inducing signal but a single isolated cell of the same type cannot. Eicosanoids are signaling molecules that often act in an autocrine mode in mature mammals. Eg. Prostaglandins These poly unsaturated fatty-acid derivatives are continuously synthesized in the plasma membrane and released to the cell exterior, where they are rapidly degraded by enzymes in extracellular fluid. When cells are activated by tissue damage or by some types of chemical signals, the rate of eicosanoid synthesis is increased; the resulting increase in the local level of eicosanoid influences both the cells that make it and their immediate neighbors. 11  Autocrine signaling can coordinate decision by groups of identical cells “Community effect” in early development In tumor biology---cancer cells stimulate their own proliferation 12 13 OVERVIEW: Types of signaling 14  Steps in Indirect Signaling Three steps are involved: 1. Release of chemical messenger from the signaling cell 2. Transport of the messenger through the extracellular environment to the target cell 3. Communication of the signal to the target cell 15  Indirect Signaling Hydrophilic messengers dissolve in aqueous solutions like extracellular fluid and blood Hydrophobic messengers bind to carrier proteins in the blood Carrier protein – help hydrophobic messengers dissolve in aqueous solutions 16 Indirect Signaling 17  Chemical Messengers Two main types 1.Hydrophilic 2.Hydrophobic 18  Ligand-Receptor Interactions Receptors bind to only one messenger or class of messengers. This property is called specificity. The strength of the binding between a messenger and its receptor is called affinity. A single messenger can bind to more than one receptor and have different affinities for each one. Only the correctly shaped ligand (natural ligand) can bind to the receptor Binding is done by non-covalent interactions (H-bonding, hydrophobic, Van der waals and electrostatic interactions.) Ligand mimics (e.g., drugs and poisons) Agonists – activate receptors Antagonists – block receptors 19  Ligand-Receptor Dynamics L+R LR →response More free ligand (L) or receptors (R) will increase the response Receptors can become saturated 20  The more receptors the target cell has the greater the response. Cell can up-regulate or increase the number of receptors when messenger concentration is low or down-regulate or decrease receptors when messenger concentration is high. 21  The greater the affinity of the receptor for the messenger the greater the response. 22  Down-regulation - when excessive numbers of transmitter molecules are available to the receptor over a period of time, a decrease in the receptor sites can be counted. This is called down-regulation. It accounts for tolerance. e.g., when heroin consistently occupies opiod binding sites. The desensitized post-synaptic neuron will not respond to average amounts of heroine, but increased amounts are required to obtain and opiod effect. This in turn induces greater levels of tolerance. 23  Up-regulation - the reverse occurs when decreased number of transmitter molecules are available at post-synaptic receptors. This leads to up-regulation with increased number of sensitized receptors. Such a condition would occur during a receptor blockade. e.g., the anti-psychotic medication thorazine blocks the dopamine receptor inducing up-regulation. As a result, additional dopamine receptors appear on the post-synaptic membrane. At this point the post-synaptic neuron is hypersensitive, and if the blockade is ended, even average amounts of dopamine can cause the movement disorder: Tardive Dyskinesia. This condition may be treated by administration of dopamine agonists or by reestablishing the dopamine blockade. Alcohol withdrawal and NMDA receptors- Chronic alcohol exposure alters NMDA receptors which leads to its upregulation. This increase in receptors is dangerous when ethanol consumption is stopped. Leads to withdrawal symptoms like tremors, agitation, hallucinations, seizures, tachycardia and hypertension. 24  Signal Transducing Systems Receptor-ligand binding leads to a complex intracellular response 25 Types of Receptors 26  Ligand-Gated Ion Channels Ligand binds to receptor Receptor changes shape opening a channel Ions move across the membrane Concentration and electrical gradients dictate the direction of ion movement Movement of ions change ion concentrations which alters the membrane potential 27  Intracellular Receptors Regulate the transcription of target genes by binding to specific DNA sequences, and increasing or decreasing mRNA production 28  Receptor Enzymes When activated by a ligand the catalytic domain starts a phosphorylation cascade Named based on the reaction catalyzed 29  G-Protein-Coupled Receptors Transmembrane protein that interacts with intracellular G- proteins G-proteins – named for their ability to bind guanosine nucleotides Activate second messengers 30  Nervous System Specialized collection of cells that can carry signals across long distances Neurons allow electrical signals to be propagated across long distances within a single cell Synapse – region in between two neurons or a neuron and other target cells Gap junctions Chemical: neurotransmitters 31  Endocrine System Sends chemicals (hormones) through the blood Produced by endocrine glands Other chemicals can act as hormones, e.g., neurohormones Non-endocrine organs can also produce hormones, e.g., heart Three types of hormones Peptides Steroids Amines 32 Three Types of Hormones 33 Systems for Cell Signaling 34 Second Messengers NON PROTEIN ONLY 35 NUCLEAR RECEPTORS 36  NUCLEAR RECEPTORS Nuclear receptors are ligand-activated gene regulatory proteins LIGANDS 37  NUCLEAR RECEPTORS Nuclear receptors have the ability to directly bind to DNA and regulate the expression of adjacent genes, hence these receptors are classified as transcription factors. The regulation of gene expression by nuclear receptors only happens when a ligand is present. Ligand binding to a nuclear receptor results in a conformational change in the receptor which in turn activates the receptor resulting in up- regulation of gene expression. Consequently nuclear receptors play key roles in both embryonic development and adult homeostasis. Nuclear receptors may be classified either according to mechanism or homology. 38  Nuclear Receptors 1. Type I a) glucocorticoid receptor i) reside in cytoplasm ii) migrates to nucleus when bound to hormone 2. Type II a) thyroid hormone receptor i) reside in nucleus ii) binding in the absence of hormone can repress transcription; binding with hormone stimulates transcription 3. Type III 4. Type IV 39 NUCLEAR RECEPTORS - Type I 40  Ligand binding to type I nuclear receptors in the cytosol results in the dissociation of heat shock proteins, homo-dimerization, translocation from the cytoplasm into the cell nucleus, and binding to specific sequences of DNA known as hormone response elements (HRE's). Type I nuclear receptors bind to HREs consisting of two half sites separated by a variable length of DNA and the second half site has a sequence inverted from the first (inverted repeat). Type I nuclear receptors include the androgen receptor, estrogen receptors, glucocorticoid receptor and progesterone receptor. The nuclear receptor/DNA complex then recruits other proteins which transcribe DNA downstream from the HRE into messenger RNA and eventually protein which causes a change in cell function. 41  Type II NUCLEAR RECEPTORS Type II receptors, in contrast to type I, are retained in the nucleus regardless of the ligand binding status and in addition bind as hetero-dimers to DNA. In the absence of ligand, type II nuclear receptors are often complexed with corepressor proteins. Ligand binding to the nuclear receptor causes dissociation of corepressor and recruitment of coactivator proteins. Additional proteins including RNA polymerase are then recruited to the NR/DNA complex which transcribe DNA into messenger RNA. Type I nuclear receptors include the retinoic acid receptor, retinoid X receptor and thyroid hormone receptor. 42 43 NUCLEAR RECEPTORS type II Ligand-binding domain 44  Type III and IV NUCLEAR RECEPTORS Type III nuclear receptors are similar to type I receptors in that both classes bind to DNA as homodimers. However, they bind to direct repeat instead of inverted repeat HREs. Type IV receptors instead bind as monomers to half-site HREs. 45  FXR-Farnesyl –Bile acids PPAR-Fibrates Orphan receptors - ligands CAR-Constitutive androstane – Xenobiotics still unknown. PXR- Pregnane -Xenobiotics 46  Plasma Membrane Receptors Different sizes and shapes Different isoforms within the family arose by gene duplication and divergence Selective expression decides physiology of the cell Ligand binding or physical activity (light, pressure, temperature, voltage) activate the receptor 47  Membrane Receptor Classes ▪ About 20 families so far ▪ Based on similarities in structure, ligand binding and signal transduction strategies ▪ Membrane Receptors include: - Ligand- gated channel - Receptor enzymes - G-protein-coupled - Integrin 48 Signal Transduction : The Process 49 For the receiving cell, there are three stages in the signaling process: Reception, Transduction and Cell Response. 50 Cell Surface Receptors May work both fast and slow Always use “second messengers” 51 52 53  Phosphatase Kinase Phosphorylase Phosphodiesterase Cyclase 54  APhosphatase phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol (converts cATP or cGTP to cADP or cGDP ). AKinase kinase is a type of phosphotransferase that transfers a phosphate group from ATP to a substrate. APhosphorylase phosphorylase is a type of phosphotransferase that catalyzes the addition of a phosphate group from an inorganic phosphate to a substrate. APhosphodiesterase phosphodiesterase is an enzyme that breaks a phosphodiester bond (converts cAMP or cGMP to AMP or GMP) ACyclase cyclase is an enzyme that catalyzes a chemical reaction to form a cyclic compound (converts AMP or GMP to cAMP or cGMP). 55 THE G PROTEIN-COUPLED RECEPTOR (GPCR) SUPERFAMILY 56  The GPCR Superfamily With more than 800 members, G-protein-coupled receptors (GPCRs) represent the largest family of cell-surface molecules involved in signal transmission, accounting for >2% of the total genes encoded by the human genome. These receptors control key physiological functions, including neurotransmission, hormone and enzyme release from endocrine and exocrine glands, immune responses, cardiac- and smooth-muscle contraction and blood pressure regulation. Their diverse ligands include hormones, neurotransmitters, opium derivatives, chemoattractants, odorants, tastants and photons. Very fast response. 57 G- Protein: G proteins (guanine nucleotide-binding proteins) are a family of proteins involved in transmitting chemical signals outside the cell, and causing changes inside the cell. They communicate signals from many hormones, neurotransmitters, and other signaling factors. Types of G protein: G protein can refer to two distinct families of proteins. Heterotrimeric G proteins: sometimes also known as the large G proteins that are activated by G protein-coupled receptors and made up of alpha (α), beta (β), and gamma (γ) subunits. Small G proteins: They are proteins of 20-25kDa The intracellular that belong to the Ras superfamily of small domain of the GPCR GTPases. These proteins are homologous to the is coupled to a alpha (α) subunit found in heterotrimers, and are heterotrimeric G- in fact monomeric. However, they also bind GTP protein. and GDP and are involved in signal transduction. 58  GPCRs are also called seven-transmembrane receptors (7TM receptors) 59  Rhodopsin was the first of these to have its 7-helix structure confirmed by X-ray crystallography. Dysfunction in a GPCR contributes to some of the most prevalent human diseases. GPCRs represent the target, directly or indirectly, of 50–60% of all current therapeutic agents. 60 61  When ligands bind a GPCR, the GPCR acquires GEF (guanine nucleotide exchange factor) ability, which activates the G-protein by exchanging the GDP on the alpha subunit to GTP. When bound to GDP, the complex is functionally inactive, with the G-α subunit remaining tightly associated with the other subunits of the GPCR complex. Thus, when the Gα subunit is bound to GDP it is “OFF”; when it is bound to GTP it is “ON” The G-α subunit contains a GTPase domain, which is capable of hydrolyzing GTP to GDP. 62  G-protein-coupled Receptor Effector + Ligand a a a GTP GTP -GDP b g GDP b g +GTP G protein G protein Second Physiological response messenger When the extracellular domain binds to the ligand, it causes a conformational change relayed through the transmembrane spans to the intracellular domain. Juang RH (2007) BCbasics The conformational change causes the Gα subunit to release GDP and bind to GTP thereby activating both the Gα and Gβ/Gγ subunits 63  Pathways after activation of G-protein Specific targets for activated G proteins include various enzymes that produce second messengers, as well as certain ion channels that allow ions to act as second messengers. The pathways downstream include: 1. The cAMP-PKA pathway cAMP is the second messenger, Adenylyl cyclase is the effector 2. The iP3-DAG pathway Both iP3 and DAG are second messengers and also Ca2+, Phospholipase C is the effector 3. The cGMP pathway cGMP is the second messenger, cGMP phosphodiesterase is the effector 64  Heterotrimeric G proteins are of five types - Gs, Gq, Gtα, Gi, and G12/13 This classification is based on the Gα subunits and the effectors to which they couple. The particular response produced by an activated GPCR depends on the type of G protein with which it interacts, although some GPCRs can interact with different G proteins and trigger more than one physiologic response. Gs family members stimulate adenylyl cyclase. Adenylyl cyclase is activated by GTP-bound Gs subunits. Gi , Activated Gi subunits function by inhibiting adenylyl cyclase 65 1) The cAMP – PKA pathway Regulation of Protein Kinase A 1)Adenylyl cyclase is the effector that is activated by Gs-α subunit. It converts AMP into cAMP. 2)Most effects of cAMP in animal cells are mediated by the action of PKA. 3)cAMP binds to the regulatory subunits, leading to their dissociation from the catalytic subunits. 4)The free catalytic subunits are then enzymatically active and able to phosphorylate Ser and Thr residues on their target proteins. 66 Regulation of Glycogen Metabolism by Protein Kinase A In the regulation of glycogen metabolism, protein kinase A phosphorylates two key target enzymes- Phosphorylase kinase and Glycogen synthase. Elevation of cAMP and activation of protein kinase A thus blocks further glycogen synthesis at the same time as it stimulates glycogen breakdown. 67 Regulation of Protein Phosphorylation by Protein Kinase A and Protein Phosphatase 1 It is important to recognize that protein kinase A do not function in isolation. The serine residues of proteins that are phosphorylated by protein kinase A are usually dephosphorylated by the action of a phosphatase called protein phosphatase I. 68 The chain of reactions leading from epinephrine receptor to glycogen phosphorylase provides a good illustration of signal amplification during intracellular signal transduction. 69  Cyclic AMP-Inducible Gene Expression In many animal cells, increase in cAMP activates the transcription of specific target genes that contain a regulatory sequence called the cAMP response element (CRE) CRE binding protein cAMP response element 70 71 2.) The IP3- DAG pathway Gq family members contain Gα subunits that activate PLC-β. PLC-β hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol trisphosphate(IP3) and diacylglycerol(DAG). PLC-β is stimulated by G proteins while PLC-γ by tyrosine kinase. 72 Hydrolysis of PIP2 One of the most widespread pathways of intracellular signaling is based on the second messengers derived form PIP2 73 Phospholipase targets 74 Of the two 2nd messengers produced, one is hydrophilic - IP3, and one is hydrophobic - DAG. The effect of DAG is to sequester protein kinase C to the membrane and stimulate its activity. The effect of IP3 is to stimulate Ca++ release from intracellular stores in the SER and mitochondria. The released Ca++ has a variety of effects one of which is the stimulation of protein kinase C. 75  Ca2+ Mobilization by IP3 IP3 acts to release Ca2+ from the ER by binding to receptors that are ligand-gated Ca2+ channel in most cells, the transient increase in intracellular Ca2+ resulting from production of IP3 triggers a more sustained increase caused by the entry of extracellular Ca2+ through channels in the plasma membrane. This entry of Ca2+ from outside the cell serves both to prolong the signal initiated by release of ligand-gated Ca2+ Ca2+ from the ER and to allow channel the stores of Ca2+ within the ER to be replenished (ER Ca2+ outside). 76  Action of Calcium After influx, Ca2+ levels increase to about 1 μM, which affects the activities of a variety of target proteins, including protein kinases and phosphatases. Ca2+ can readily bind to proteins and cause conformational changes. Changes in intracellular Ca2+ are detected by Ca2+-binding proteins that regulate a variety of Ca2+-dependent enzymes like Calmodulin. 77 78  ADRENERGIC RECEPTORS (EPINEPHRINE) The α and the β adrenergic receptors: ○ Occur on separate tissues in mammals ○ Respond differently to catecholamines ○ Almost opposite effects The mechanism of adrenergic receptors: Adrenaline or noradrenaline are receptor ligands to either α1, α2 or β- adrenergic receptors. α1 couples to Gq, which results in increased intracellular Ca2+ which results in smooth muscle contraction. α2, couples to Gi, which causes a decrease of cAMP activity, resulting in smooth muscle contraction. β receptors couple to Gs, and increases intracellular cAMP activity, resulting in heart muscle contraction, smooth muscle relaxation and glycogenolysis. 79 80 81 82 83 How to turn OFF the signal? 1. Gα hydrolyzes GTP to GDP + Pi. (GTPase). The presence of GDP on Gα causes it to rebind to the inhibitory βγ complex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterases catalyze hydrolysis of cAMP → AMP. 3. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A. 84 4. Receptor desensitization varies with the hormone. In some cases the C-terminal of the activated receptor is phosphorylated via a G-protein Receptor Kinase. The phosphorylated receptor then may bind to a protein β- arrestin. β-Arrestin promotes removal of the receptor from the membrane by clathrin-mediated endocytosis. β-Arrestin may also bind a cytosolic Phosphodiesterase, bringing this enzyme close to where cAMP is being produced, contributing to signal turnoff. 85  Rhodopsin Signaling 1. Light Hyperpolarizes Rod Cells of the Vertebrate Eye 11-cis-retinal 2. Light Triggers Conformational Changes in the Receptor Rhodopsin 3. Excited Rhodopsin Acts through the G Protein Transducin (Gtα) {a variant of Giα} to Reduce the cGMP Concentration 4. Amplification of the Visual Signal Occurs 5. The Visual Signal Is Quickly Terminated 6. Rhodopsin Is Desensitized by Phosphorylation 86 87 G protein signaling can be blocked by bacterial toxins 88 89 90 91 94. In cells having G protein coupled receptor, inhibition of protein kinase A by siRNA technology led to diminished transcription of androgen binding protein (ABP) and CREB protein. Addition of cAMP, which is a second messenger, will lead to 1. increased transcription of ABP 2. increased phosphorylation of CREB protein. 3. no change in transcription level 4. increased GTPase activity of G α subunit. 92 93 94 95 Receptors that are enzymes: RECEPTOR TYROSINE KINASES 96  Receptors that are enzymes: Receptor Tyrosine Kinases Receptor tyrosine kinases phosphorylate tyrosine residues of cytoplasmic signaling molecules (also tyrosine kinases). 60 RTKs are encoded by the human genome. Involved in the regulation of growth, division, differentiation, survival, and attachment to extracellular matrix. Expression of mutant RTKs that are constitutively active leads to cancer. 97 Ligands of receptor tyrosine kinases (RTK)- GROWTH FACTORS What are Growth Factors? Ligands which bind enzyme linked receptors and signal diverse cellular responses including: Proliferation Differentiation Growth Survival Angiogenesis Can signal to multiple cell types or be specific 98 Ligands of receptor tyrosine kinases (RTK)- GROWTH FACTORS a) Nerve growth factor (NGF) b) Platelet-derived growth factor (PDGF) c) Fibroblast growth factor (FGF) d) Epidermal growth factor (EGF) e) Insulin and insulin-like GF (IGF-1) f) Ephrins (Eph) g) Vascular endothelial factor (VEGF) Subfamilies of receptor tyrosine kinases (RTK) 99 100 NAMES OF SOME RECEPTORS Guide cell/axon migration angiogenesis 101  Receptor activation Ligand binding results in the conformational change and dimerization of the extracellular ligand –binding domains of a pair of receptors. Two mechanisms: 1. Ligand-mediated dimerization 2. Receptor mediated dimerization 102 103 104 Exception: Activation of Insulin receptor Some RTK are already dimers (insulin receptor) but ligand binding is necessary for activity 105  Transcription factor Adaptor protein Docking protein Diversity of signalling proteins Signalling enzymes 106 ADAPTER PROTEINS Adapter proteins do not contain catalytic domains, but they serve to link phosphorylated tyrosine receptors to other effector proteins. E.g. Shc (SH2 domain and collagen-like), FRS2 (FGF receptor substrate 2), Grb2 (growth factor receptor bound 2), p85 (PI 3-kinase), mSOS (mammalian son of sevenless), etc. Phosphorylated tyrosines act as docking sites for Src- Homology 2 (SH2) domain containing proteins or PhosphoTyrosine Binding (PTB) domain containing proteins. These 2 are called protein interaction domains. SH3 and PH are other known domains. SH2-domains are peptide segments of about 100 amino acids that are capable of binding to phosphorylated tyrosines. Several signal transducing molecules contain SH2-domains. PTB domains consist of 100-150 amino acids and are found in signaling molecules like insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). 107 Phosphorylated tyrosine serves as docking sites for adapter proteins with SH2 or PTB domains 108 109  Activation of the ERK MAP Kinases 1) The MAP kinase pathway refers to a cascade of protein kinases that are highly conserved in evolution and play central roles in signal transduction in all eukaryotic cells. 2) In higher eukaryotes, MAP kinases are ubiquitous regulators of cell growth and differentiation. 110 Regulation of Ras Proteins Interest in Ras intensified in 1982, when mutations in ras genes were first implicated in the development of human Guanine nucleotide cancers. exchange factor (GEF) The mutations of ras genes in human cancers have the effect of inhibiting GTP SOS hydrolysis by the Ras proteins. These mutated Ras proteins then remain continuously activated (constitutively active), driving the unregulated proliferation of cancer cells even in the GTPase- activating absence of growth factor proteins (GAP) stimulation. 111 Guanine Exchange Factor (GEF), GTPase activating protein (GAP) Guanine dissociation inhibitor (GDI) 112  Signal transduction cascades Signal The successive phosphorylation/activation p of multiple kinases results in a cascade of KINASE #1 phosphorylation/activation This cascade is frequently called a signal- transduction cascade p This cascade eventually leads to a specific KINASE #2 cellular response e.g. changes in cell physiology and/or patterns of gene expression p RTK pathways are involved in regulation of KINASE #3 cell proliferation and differentiation, promotion of cell survival, and modulation of cellular metabolism p TARGET EFFECT 113  Ras – MAPK (Mitogen Activated Protein Kinase) pathways MAP kinases are actually a family of protein kinases that are widely distributed and are found in all eukaryotes. The mitogen activated protein kinase pathways (MAPKs) represent highly conserved cascades activated in response to effectors downstream of receptor tyrosine kinases (RTKs) or in some cases G- protein coupled receptors (GPCRs), and in response to inflammatory cytokines and stress. Together, they control many cellular processes. The hallmark of MAPK pathways is the shared architecture of a three- tier signaling core, namely, MAPKKK/MAPKK/MAPK. 114 115  The downstream tier and main effectors MAPKs are activated by phosphorylation of threonine and tyrosine residues within a conserved TxY motif in the activation loop by dual specificity kinases MAPKKs of the second tier, in turn activated by MAPKKKs serine/threonine kinases of the third tier. The MAPKs are serine/threonine kinases whose targets include transcription factors and regulators, other nuclear or cytoplasmic proteins, kinases collectively known as MAPKAPK that further transmit the signal. Docking interactions between members of the three tiers and with substrates and interactions with scaffold proteins assure the organization of distinct MAPK pathways and regulate the specificity of their signaling. Phosphatases and other regulatory proteins along with feedback loops act to shape these signaling cascades. 116  Overview of MAPK activation and signaling cascade Activation of a receptor tyrosine kinase (RTK) by its appropriate growth factor recruit SH2 domains of Grb2. Guanine nucleotide exchange factors (GEF) such as SOS are localized to the membrane by Grb2, which then stimulate Ras to exchange GDP for GTP. Ras signaling is terminated when GTPase- activating proteins such as p120 and NF-1 stimulate Ras to hydrolyze GTP to GDP. The activated Ras recruits and activates Raf at the plasma membrane, which then phosphorylates MEK in the cytoplasm. Activated MEK subsequently phosphorylates ERK, which translocates to the nucleus where it activates multiple transcription factors, which ultimately results in effector protein synthesis causing changes in cell proliferation and cell survival. 117 Activation of Phospholipase C by Tyrosine Kinases PIP2 is activated downstream of both G protein- coupled receptors and protein- tyrosine kinases. PLC-β is stimulated by G proteins, PLC-γ by tyrosine kinase. 118 119 EGF Signal Transduction 120  Insulin signaling 1. Binding → Dimerization → Phosphorylation 2. Binding and phosphorylation of IRS1,2 3. Binding and activation of PI-3-K by IRS 4. Formation of PIP3 5. Activation of PIP3 dependent protein kinase 1 (PDK1) 6. Recruitment and Activation of Akt 7. Kinase cascade 8. GLUT4 transporter translocation, inactivation of GSK3, activation of glycogen synthase, etc. 121  The PI3-Kinase/Akt/mTOR pathway The kinases that convert PI (phosphatidylinositol) to PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions 4 & 5 of the inositol ring. PI 3-Kinases catalyze phosphorylation of PI at the 3 position of the inositol ring. The binding of insulin to its cell-surface receptor promotes its tyrosine kinase activity, the recruitment of insulin receptor substrate 1 (IRS1), the production of phosphatidylinositol (3,4,5)-triphosphate (PIP3) through the activation of PI3K, and the recruitment and activation of AKT at the plasma membrane. The PI3K/Akt/mTOR pathway plays a key role in cell signaling, regulating proliferation, survival and differentiation phosphatidylinositol 3,4,5-triphosphate 122 (PIP3) Activation of the Akt protein kinase (PKB) by PIP3 PIP3 functions as a distinct second messenger. A key target of PIP3, which is critical for signaling cell survival, is a protein- serine/threonine kinase called Akt. Akt resides in the cytoplasm in the inactive state. PIP3 binds to a domain of Akt known as the pleckstrin homology domain (PH). This interaction recruits Akt to the inner face of the plasma membrane, where it is phosphorylated and activated by Phosphoinositide dependent kinase 1 (PDK1) that also contains a PH domain. 123 Akt Akt Akt Akt 124 Activation of mTOR by Akt Akt indirectly activates (not phosphorylates) mTOR which lies at the heart of growth regulatory pathways. mTOR promotes cell growth by: - activating S6 kinase (an activator of translation) - activating PKC (so turning on many synthetic and secretory pathways) - inhibiting p21 (so releasing cells from G1 arrest) - inhibiting GSK3β (with similar effect, since GSK3β targets cyclin D for proteolysis) Akt also phosphorylates Bad (a pro-apoptotic protein,which in its non-phosphorylated state, promotes apoptosis) which sequesters it and keeps it out of action, promoting cell survival. 125 126 127  NET Questions Proteins with cytoplasmic domains having tyrosine kinase activity do NOT act as receptors for: A. Epidermal growth factor (EGF) B. Platelet-derived growth factor (PDGF) C.Insulin D.Transferrin 128 129 130 131 Receptors that are enzymes: GUANYLYL CYCLASE RECEPTOR 132  Receptors that are enzymes: Guanylyl cyclase receptor ❑ Another receptor with intrinsic enzymatic activity (guanylyl cyclase) ❑ Binding of the ligand activates the enzymatic activity of the receptor molecule ❑ Receptor for Atrial Natriuretic Factor and other peptides ❑ Receptor catalyzes formation of cGMP from GTP Cyclic GMP ❑ cGMP regulates ⮚ cGMP gated ion channels ⮚ cGMP activated protein kinases ⮚ cGMP-phosphodiesterases 133 134 135 136 137 Receptors that are enzymes: Guanyl cyclase receptor Ligand binding to receptor elevates Ca+2 levels (Ca+2 channel or IP3 mechanism) Which stimulates the activity of nitric oxide synthtase (NOSase) resulting in the production of NO from arginine. NO diffuses to soluble guanylyl cyclase and stimulates its activity resulting in the production of cGMP. The increase in intracellular cGMP then acts via standard mechanisms The interesting thing about this mechanism is that NO is lipid soluble and can cross membranes and stimulate neighboring cells. This mechanism means that NO is really an autocrine/paracrine factor rather than being a second messenger. 138 Receptors that are enzymes: RECEPTOR PROTEIN SERINE/THREONINE KINASE 139 140  LIGANDS Transforming growth factor - β superfamily TGF-β proteins Bone Morphogenetic Proteins (BMPs), Activins, Inhibins, Nodal, Lefty, Mülllerian Inhibiting Substance (MIS) Growth Differentiation Factors (GDFs) Glial-derived Neurotrophic Factors (GDNFs) Ligands of the TGF-beta superfamily form dimers that bind to heterodimeric receptor complexes. 141  TGF-β superfamily -Serine threonine kinase Signal proteins of the TGF-β superfamily act through receptor serine/threonine kinases and Smads TGFβ receptors are single pass serine/threonine kinase receptors. They exist in several different isoforms that can be homo- or heterodimeric. The number of characterized ligands in the TGFβ superfamily far exceeds the number of known receptors, suggesting the promiscuity that exists between the ligand and receptor interactions. TGF-β/Smad pathway has a more direct connection between growth factor receptors and transcription factors. 142  SMADs There are three distinct sub-types of Smads: 1. Receptor-regulated Smads (R-Smads), 2. Common partner/ mediator Smads (Co-Smads), and 3. Inhibitory Smads (I-Smads). The R-Smads consist of Smad1, Smad2, Smad3, Smad5 and Smad8/9, and are involved in direct signaling from the TGF- B receptor. Smad4 is the only known human Co-Smad, and has the role of partnering with R-Smads to recruit co-regulators to the complex. Smad6 and Smad7 are I-Smads that can bind to type I receptors and prevent the phosphorylation of the R-SMADs. 143 TGF-β superfamily -Serine threonine kinase 144 145 146 Receptors that are not enzymes but linked to enzymes CYTOKINE RECEPTORS, TOLL LIKE RECEPTORS 147 Receptors that are not enzymes but linked to enzymes : 1. Cytokine Receptors 148  LIGANDS The term "cytokine" has been used to refer to the immunomodulating agents, such as interleukins and interferons. lymphokines, interleukins, chemokines 149 Cytokine receptors function as oligomeric complexes consisting of typically two to four receptor chains that may be the same or different. In single subunit receptors the subunits fulfill the dual role of binding to cytokines and signaling. – growth hormone (GH), – erythropoietin (EPO), – granulocyte colony-stimulating factor (G-CSF ), and – thrombopoietin (TPO). In multi-subunit receptors the different subunits may perform specialized functions such as ligand-binding or signal transduction. – granulocyte-macrophage CSF (GM-CSF), – interleukin-3 (IL-3), and – IL-5 and IL-6 – CNTF receptor (CNTFR) – IL-2 receptor (IL-2R) 150  Receptors linked to enzymes Phosphorylation of an associated enzyme. Receptors for a variety of cytokines and growth factors do not have their own protein kinase activity but are associated on the intracellular side with tyrosine kinases or Ser- Thr kinases. One tyrosine kinase is called JAK which can activate downstream effectors that include the STAT proteins - the JAK-STAT pathway 151  Receptors linked to enzymes The JAK/STAT Pathway In the JAK/STAT pathway, protein- tyrosine phosphorylation directly affects transcription factor localization and function. JAK - Just Another The STAT proteins are transcription factors that contain SH2 domains that Kinase mediate their binding to phosphotyrosine - containing sequences. In unstimulated cells, STAT proteins are inactive in the cytosol. Stimulation of cytokine receptors leads to the binding of STAT proteins where they STAT - Signal are phosphorylated by the receptor- Transducers associated JAK protein-tyrosine kinases. and Activators The phosphorylated STAT proteins then of dimerize and translocate to the Transcription nucleus where they activate the transcription of target genes. 152 153 154  2. Toll-Like Receptors Toll-Like (TLRs) TLRs are single-pass membrane- spanning receptors usually expressed on sentinel cells such as macrophages and dendritic cells. The innate immune system employs germline-encoded pattern-recognition receptors (PRRs) for the initial detection of microbes. PRRs recognize microbe-specific molecular signatures known as pathogen- associated molecular patterns (PAMPs) and self-derived molecules derived from damaged cells, referred as damage- associated molecules patterns (DAMPs). 155 156  TLR1, TLR2, TLR4, TLR5, and TLR6 are located on the cell membrane, TLR3, TLR7, TLR8, and TLR9 are located in intracellular vesicles (because they are sensors of nucleic acids). 157 158 Developmental pathways WNT, HEDGEHOG AND NOTCH PATHWAYS 159 The Hedgehog and Wnt pathways are closely connected signaling systems that play key roles in determining cell fate during embryonic development. 1. Hedgehog Pathway: The hedgehog genes encode secreted proteins that are modified by the addition of lipids. Hedgehog proteins: Hh Fruit fly larva lacking the Hh gene are said to resemble hedgehogs. 160 The Hh/Ci signalling pathway in Drosophila mainly includes: – The Hh ligand, – Its twelve-pass transmembrane protein receptor Patched (Ptc), – The seven-pass transmembrane protein Smoothened (Smo), It is a G-protein-coupled receptor-like protein – Cytoplasmic proteins involved in the Hh signalling protein complex, including: Fused kinase, Costal-2 (Cos2), GSK3 beta, PKA, Fu suppressor protein (SuFu) – The transcription factor Cubitus interruptus (Ci) 161  In the absence of Hedgehog, Ci is maintained in a complex with a protein kinase called Fused and a kinesin-related protein called Coastal-2, which anchors the complex to microtubules. Within this complex, Ci is either completely degraded or cleaved to generate a transcriptional repressor (Ci75). Hedgehog binds to Patched, which acts as a negative regulator of Smoothened. 162  The binding of Hedgehog to Patched allows Smoothened to propagate an intracellular signal, leading to activation of a zinc finger transcription factor called Cubitus interruptus (Ci) in Drosophila (Gli in mammals). Hedgehog signaling promotes the interaction of Smoothened with Coastal-2, leading to the release of full-length Ci (Cil55), which is then able to translocate to the nucleus and activate transcription of its target genes. 163 Schematic representation of the Hedgehog (Hh) signalling pathway in Drosophila. 164  Patched (PTC), Smoothened (Smo) and Cubitus interruptus (Ci) (Left) Ptc represses Smo, preventing the activation of Hedgehog signalling via proteolytic cleavage of Ci. Cleavage results in a repressor form of Ci, which enters the nucleus and inhibits Hedgehog target gene expression. (right) In the presence of Hedgehog (Hh) the inhibitory effects of Ptc on Smo are relieved. Smo becomes phosphorylated by PKA and CK1 and PKA, CK1 and GSK3 are released from Cos2 (Costal2), preventing the cleavage of Ci. Full length Ci is no longer inhibited by Sufu and enters the nucleus to induce the 165 transcription of Hedgehog target genes.  In higher organisms, the core constituents of Hh signalling are more complex. Comprises of: ❖ Three Hh ligands, Sonic hedgehog (Shh), Desert hedgehog (Dhh) and Indian hedgehog (Ihh); ❖ Two twelve pass transmembrane receptors, Patched1 (PTCH1) and Patched2 (PTCH2); ❖ Smo ❖ Three transcription factors, GLI (glioma-associated oncogene family zinc finger) GLI1, GLI2 and GLI3 (Gli1 is involved in transcriptional activation, while Gli2 and Gli3 can both activate and inhibit transcription) 166 167 2. Wnt Pathway: Wnt signaling is one of the key cascades regulating development and has also been tightly associated with cancer. The name ‘Wnt’ = Wingless gene discovered in Drosophila + Integration gene discovered in mice The Wnt pathway is commonly divided into β-catenin dependent (canonical) and independent (non-canonical) signaling. – Canonical or Wnt- β catenin pathway. (β-catenin dependent) – Non-canonical or Wnt-Calcium pathway (β-catenin independent) 168  2. Wnt Pathway: The Wnt proteins are a family of secreted growth factors that bind to Frizzled family receptors and co- receptor LRP. Signaling from LRP and Frizzled leads to activation of a cytoplasmic protein called Dishevelled, and inhibition of a complex of the proteins Axin, APC, and the protein kinase GSK-β. Within this complex, GSK- β phosphorylates β -catenin, leading to its ubiquitination and degradation, so Wnt signaling results in increased β -catenin levels. 169 170  In canonical Wnt signaling, absence of Wnt ligands leads to phosphorylation of β-catenin by the destruction complex, which contains the scaffold protein Axin, APC and the kinases GSK3β and casein kinase (CK1α). In this state, β-catenin is phosphorylated by GSK3β, ubiquitinated by β- TrCP200 and targeted for proteasomal degradation. In the absence of nuclear β-catenin, a repressive complex containing TCF/LEF and transducing-like enhancer protein (TLE/Groucho) recruits HDACs to repress target genes. The canonical pathway is activated upon binding of secreted Wnt ligands to Fzd receptors and LRP co-receptors. LRP receptors are then phosphorylated by CK1α and GSK3β, which recruits Dishevelled (Dvl) proteins to the plasma membrane where they polymerize and are activated. The Dvl polymers inactivate the destruction complex which results in stabilization and accumulation of β-catenin which then translocates into the nucleus. There, β-catenin forms an active complex with LEF (lymphoid enhancer factor) and TCF (T-cell factor) proteins by displacing TLE/Groucho complexes and recruitment of histone modifying co-activators. 171 172 3. Notch Pathway: Notch signaling involves direct cell-cell interactions during development. Notch signaling promotes proliferative signaling during neurogenesis, and its activity is inhibited by Numb to promote neural differentiation. Notch is a large protein with a single transmembrane domain that serves as a receptor for transmembrane proteins that are members of the DSL (Delta/Serrate/LAG-2) family of proteins on the surface of adjacent cells. In Drosophila, there are two ligands named Delta and Serrate. In mammals, the corresponding names are Delta-like and Jagged as well as other ligands, such as F3/contactin. Like the Wnt signaling pathway, the Notch target genes include genes encoding other transcriptional regulatory proteins, which act to determine cell fate. 173  Notch receptor performs direct cell- cell signaling by binding to transmembrane proteins (e.g., Delta) on neighboring cells. The binding of Delta leads to proteolytic cleavage of Notch by y- secretase. The intracellular domain of Notch(NICD) is then translocated into the nucleus. The Notch intracellular domain then interacts with a transcription factor (called Su[H] in Drosophila, or CSL in mammals) and converts it from a repressor to an activator of its 174 target genes. 175 176 177 178 179 180 (i) FGF (a) Patched (ii) Hedgehog (b) Frizzled (iii) Wnt (c) Receptor tyrosine kinase 1. i - c, ii - a, iii - b 2. i - a, ii - c, iii - b 3. i - b, ii - c, iii - a 4. i - c, ii - b, iii - a 181 182 183 184 185 186