Cell Signaling

Page 1: Overview of Cell Signaling

Cell signaling encompasses the fundamental mechanisms through which cells detect, transduce, and respond to environmental stimuli and intercellular cues to coordinate cellular function.

Page 2: Principles of Cell Communication

Cell communication enables both unicellular and multicellular organisms to survive, develop, obtain nutrients, reproduce, and coordinate activities within tissues. Environmental and intercellular signals can be chemical or mechanical.

In budding yeast, mating involves communication between two distinct haploid cell types ($a$ and $\alpha$). Each cell type secretes a specific peptide mating factor ($a$ factor or $\alpha$ factor) that binds to surface receptors on the opposite cell type. Factor binding induces cell fusion, producing a diploid $a/\alpha$ cell containing all genetic material from both parent cells.

Page 3: General Concepts in Signal Transduction

Signal transduction is the process by which an extracellular impulse or stimulus is converted from one physical or chemical form into another across the cell boundary. Extracellular signal molecules bind to specific cellular receptors, activating intracellular signaling pathways that ultimately modify effector proteins and alter cell behavior.

Page 4: Intercellular Signaling Ranges

Intercellular communication operates across diverse spatial scales:

• Endocrine Signaling: Endocrine cells secrete hormones into the bloodstream, distributing them systemically throughout the entire body to act on distant target cells (e.g., insulin).

• Paracrine Signaling: Signaling cells secrete local mediators into the extracellular fluid to act on neighboring cells within the immediate vicinity (e.g., growth factors).

• Neuronal (Synaptic) Signaling: Neurons propagate electrical action potentials along axons to nerve terminals, triggering the release of neurotransmitters across specialized synapses to stimulate target cells (e.g., acetylcholine).

• Contact-Dependent Signaling: Membrane-bound signal molecules on the surface of one cell interact directly with receptor proteins on an adjacent cell without requiring the secretion of a diffusible ligand.

Page 5: Synaptic Transmission Architecture

In excitatory synapses of the central nervous system, communication occurs between a presynaptic cell terminal and a postsynaptic target cell across a specialized synaptic cleft measuring $< 100\,nm$. Arrival of an action potential induces exocytosis of synaptic vesicles, discharging neurotransmitters into the cleft to bind postsynaptic receptors.

Page 6: Direct and Local Communication

Signaling can be mediated by widely circulated hormones, locally diffusing paracrine factors, synaptic neurotransmitters, or direct membrane-membrane contact between cells.

Page 7: Contact-Dependent Immune Signaling

Antigen presentation exemplifies contact-dependent signaling. Dendritic cells (DCs) and other professional antigen-presenting cells (APCs) display processed peptide antigens via major histocompatibility complex (MHC) proteins directly to T cell receptors (TCR) on $CD4^+$ or $CD8^+$ T cells. Costimulatory interactions between $CD80/86$ on APCs and $CD28$ on T cells trigger T cell clonal expansion and cytokine secretion (such as $IFN\text{-}\gamma$).

Page 8: Classes of Extracellular Signal Molecules

Extracellular signal molecules exhibit diverse biochemical structures and physiological functions:

• Hormones:

  • Epinephrine (adrenaline): Tyrosine derivative secreted by the adrenal gland; increases blood pressure, heart rate, and metabolism.

  • Cortisol: Steroid derived from cholesterol secreted by the adrenal gland; regulates carbohydrate, protein, and lipid metabolism.

  • Estradiol: Steroid derived from cholesterol secreted by the ovary; maintains female secondary sexual characteristics.

  • Testosterone: Steroid derived from cholesterol secreted by the testis; maintains male secondary sexual characteristics.

  • Insulin: Protein secreted by $\beta$ cells of the pancreas; stimulates glucose uptake and macromolecular synthesis.

  • Thyroid hormone (thyroxine): Tyrosine derivative secreted by the thyroid gland; regulates metabolic rate across multiple tissues.

• Local Mediators:

  • Epidermal growth factor (EGF): Protein; stimulates proliferation of epidermal and epithelial cells.

  • Platelet-derived growth factor (PDGF): Protein released by platelets; stimulates cell division.

  • Nerve growth factor (NGF): Protein; promotes survival and axonal outgrowth in specific neurons.

  • Histamine: Histidine derivative released by mast cells; induces vascular dilation and permeability during inflammation.

  • Nitric oxide (NO): Dissolved gas synthesized by endothelial and nerve cells; induces smooth muscle relaxation.

• Neurotransmitters:

  • Acetylcholine: Choline derivative; excitatory neurotransmitter at neuromuscular junctions and in the central nervous system.

  • $\gamma$-Aminobutyric acid (GABA): Glutamic acid derivative; primary inhibitory neurotransmitter in the central nervous system.

• Contact-Dependent Molecules:

  • Delta: Transmembrane protein; inhibits neighboring cells from adopting the identical developmental fate.

Page 9: Cellular Receptor Compartmentalization

Signal reception depends on ligand physicochemical properties:

• Cell-Surface Receptors: Bind large, hydrophilic, non-membrane-permeable signaling molecules that cannot cross the plasma membrane.

• Intracellular Receptors: Bind small, hydrophobic, membrane-permeable molecules that diffuse across the plasma membrane into the cytosol or nucleus.

Page 10: Signal Response Specificity

The cellular response to an extracellular ligand depends on the receptor subtype and the specialized intracellular machinery of the target cell. Acetylcholine binds to different receptors and triggers distinct physiological outcomes across different cell types:

• Heart Pacemaker Cell: Decreases rate of action potential firing.

• Salivary Gland Cell: Triggers exocytic secretion of saliva components.

• Skeletal Muscle Cell: Induces muscle cell contraction.

Page 11: Combinatorial Signaling and Cell Fate

Individual cells are constantly exposed to multiple extracellular cues. Specific combinations of signals instruct a cell to survive, divide, or differentiate. Deprivation of essential survival signals triggers apoptotic cell death.

Page 12: Kinetic Profiles of Signaling Responses

Cellular responses vary widely in execution speed:

• Fast Responses ($< 1\,s$ to minutes / milliseconds): Signals directly alter the function of existing proteins and cytoplasmic machinery (e.g., phosphorylation, ion channel opening, secretion, motility).

• Slow Responses (minutes to hours): Signals require altered gene expression, transcription of mRNA, and new protein synthesis (e.g., cell growth, proliferation, differentiation).

Page 13: General Principles of Cell-Surface Signaling

Cell-surface receptors receive extracellular signals that cannot cross the plasma membrane and relay the information inside through an intracellular signaling network.

Page 14: Stages of Cell Signaling

Cell signaling proceeds in three sequential stages:

1. Reception: Extracellular signal molecule binds specifically to its designated target receptor.

2. Transduction: Receptor activation initiates an intracellular relay cascade, converting the signal into different biochemical forms and amplifying it.

3. Response: Effector proteins are altered to change cellular behavior, including enzyme activation, cytoskeletal rearrangement, or gene transcription.

Page 15: Effector Proteins and Cellular Outputs

Intracellular signaling pathways converge on specific classes of effector proteins to execute distinct biological outputs:

• Metabolic Enzymes: Alter cellular metabolism.

• Cytoskeletal Proteins: Alter cell shape or cell movement.

• Transcription Regulators: Alter patterns of gene expression.

Page 16: Operational Roles of Intracellular Cascades

Intracellular signaling pathways perform several critical processing tasks:

• Relay: Pass the signal onward through the cell.

• Amplify: Generate large quantities of secondary messengers to produce a robust response from very few extracellular molecules.

• Integrate: Combine incoming signals from multiple converging pathways.

• Distribute: Direct information to multiple effector proteins, eliciting a complex response.

• Feedback: Modulate upstream signaling components through feedback loops.

Page 17: Molecular Switches in Signaling

Many key intracellular relay molecules cycle between inactive and active conformations, functioning as molecular switches to control information flow.

Page 18: Feedback Circuitry in Signal Transduction

Signaling loops employ two major feedback architectures:

• Positive Feedback: A downstream product reinforces an upstream activating component, generating an irreversible, all-or-none, explosive biological response.

• Negative Feedback: A downstream product inhibits an upstream component, generating self-limiting or oscillatory dynamics.

Page 19: Classes of Molecular Switches

Two major classes of molecular switches regulate signaling pathways:

• Protein Phosphorylation Switches:

  • Protein kinases covalently transfer a phosphate group from $ATP$ to specific amino acid side chains (Serine/Threonine kinases or Tyrosine kinases), toggling the protein to an active (or inactive) state.

  • Protein phosphatases remove the phosphate via hydrolysis ($P_i$), restoring the basal state.

• GTP-Binding Protein Switches:

  • Proteins switch to an active conformation upon binding $GTP$.

  • Intrinsic $GTPase$ activity hydrolyzes $GTP$ to $GDP$ ($P_i$ released), reverting the protein to its inactive conformation.

Page 20: Regulation of Monomeric GTPases

Monomeric GTP-binding proteins are tightly controlled by two regulatory proteins:

• Guanine Nucleotide Exchange Factors (GEFs): Promote activation by inducing the release of bound $GDP$ and uptake of $GTP$.

• GTPase-Activating Proteins (GAPs): Promote inactivation by stimulating the protein's intrinsic $GTP$ hydrolysis rate.

Page 21: Major Classes of Cell-Surface Receptors

Cell-surface receptors fall into three principal classes:

1. Ion-Channel-Coupled Receptors: Transmitter-gated ion channels that open upon ligand binding, allowing rapid ion flow across membranes to alter membrane potential in excitable cells.

2. G-Protein-Coupled Receptors (GPCRs): Receptors that activate membrane-associated heterotrimeric G proteins, which in turn regulate downstream enzymes or ion channels.

3. Enzyme-Coupled Receptors: Receptors that function directly as enzymes or recruit and activate associated intracellular enzymes upon ligand binding.

Page 22: Exogenous Compounds Acting on Cell-Surface Receptors

Numerous drugs and toxins exert their physiological effects by mimicking or blocking endogenous ligands:

• Barbiturates and Benzodiazepines (Valium, Ambien): Stimulate GABA-activated ion-channel-coupled receptors; produce sedation and relief of anxiety.

• Nicotine: Stimulates acetylcholine-activated ion-channel-coupled receptors; constricts blood vessels and elevates blood pressure.

• Morphine and Heroin: Stimulate G-protein-coupled opiate receptors; induce analgesia and euphoria.

• Curare: Blocks acetylcholine-activated ion-channel-coupled receptors; blocks neuromuscular transmission, causing paralysis.

• Strychnine: Blocks glycine-activated ion-channel-coupled receptors; blocks central nervous system inhibitory synapses, causing seizures and spasms.

• Capsaicin: Stimulates temperature-sensitive ion channels; provokes a painful burning sensation followed by desensitization.

• Menthol: Stimulates temperature-sensitive ion channels; elicits a cool sensation at moderate levels and burning pain at high doses.

Page 23: Ion-Channel-Coupled Receptor Activation

At chemical synapses, neurotransmitters released from the presynaptic nerve terminal bind transmitter-gated ion channels on the postsynaptic membrane. Channel opening drives rapid ion flux across the membrane, altering the postsynaptic membrane potential and transducing a chemical signal into an electrical impulse.

Page 24: Nicotinic Acetylcholine Receptor Structure

The nicotinic acetylcholine receptor at the neuromuscular junction is a pentameric protein complex forming a central transmembrane pore. Binding of two acetylcholine molecules to extracellular binding sites alters channel conformation, opening a hydrophobic gate. Negatively charged amino acid side chains at the mouth of the pore ensure selectivity exclusively for cations ($Na^+$ and $K^+$).

Page 25: Overview of G-Protein-Coupled Receptors

GPCRs constitute the largest and most functionally versatile family of membrane receptors in eukaryotic organisms.

Page 26: Molecular Structure of GPCRs

GPCRs share a conserved evolutionary architecture consisting of a single polypeptide chain with seven transmembrane $\alpha$ helices (seven-pass transmembrane receptors). Extracellular loops create the ligand-binding pocket for signaling molecules (such as epinephrine), while cytosolic loops engage intracellular heterotrimeric G proteins.

Page 27: Structure and Discovery of G Proteins

Discovered by Alfred G. Gilman and Martin Rodbell (1994 Nobel Prize in Physiology or Medicine), heterotrimeric G proteins consist of three subunits: $\alpha$, $\beta$, and $\gamma$. The $G\alpha$ and $G\gamma$ subunits are anchored to the cytosolic face of the plasma membrane by lipid tails. In the inactive ground state, the $G\alpha$ subunit is bound to $GDP$.

Page 28: GPCR-Mediated G Protein Activation

Extracellular ligand binding induces a conformational change in the GPCR, enabling it to act as a GEF for its cognate G protein. The $G\alpha$ subunit releases $GDP$ and binds $GTP$, causing the G protein to separate or reorient into an active $G\alpha\text{-GTP}$ monomer and an active $G\beta\gamma$ heterodimer, both of which can engage downstream effectors.

Page 29: Inactivation of G Proteins via GTP Hydrolysis

The $G\alpha$ subunit contains intrinsic $GTPase$ activity that hydrolyzes its bound $GTP$ to $GDP$ ($P_i$ released). Following hydrolysis, $G\alpha\text{-GDP}$ dissociates from its effector and reassembles with the $G\beta\gamma$ complex to regenerate the inactive G protein trimer.

Page 30: Regulation of Pacemaker K+ Channels by Gi

In cardiac pacemaker cells of the sinoatrial (SA) node, acetylcholine binding to muscarinic GPCRs activates an inhibitory G protein ($G_i$). The released, active $G\beta\gamma$ complex binds directly to the intracellular surface of $K^+$ channels, opening them. Resulting $K^+$ efflux hyperpolarizes the pacemaker cell membrane, decreasing heart rate. Inactivation occurs when $G\alpha$ hydrolyzes its bound $GTP$, prompting $G\beta\gamma$ reassociation and channel closure.

Page 31: Second Messenger Production by G Protein Effectors

Activated $G\alpha$ subunits often interact directly with membrane-bound enzymes, stimulating the rapid generation and cytosolic diffusion of small secondary messenger molecules that relay and amplify the signal.

Page 32: Cyclic AMP Synthesis and Degradation

Cyclic adenosine monophosphate ($cAMP$) is synthesized from $ATP$ by adenylyl cyclase via elimination of pyrophosphate ($PP_i$). Cyclic AMP phosphodiesterase ($PDE$) hydrolyzes the cyclic phosphodiester bond ($+ H_2O$) to form regular $AMP$. Caffeine competitively inhibits phosphodiesterase in the nervous system, maintaining elevated $cAMP$ levels.

Page 33: Dynamics of Intracellular Cyclic AMP Concentration

Resting neurons maintain a basal cytosolic $cAMP$ concentration of $5 \times 10^{-8}\,M$. Exposure to serotonin prompts a twentyfold ($20\times$) increase in $cAMP$ to $> 10^{-6}\,M$ within $50\,s$, concentrated primarily near active receptor clusters.

Page 34: Fast GPCR-cAMP Cascade: Glycogen Breakdown

During "fight-or-flight" responses, epinephrine binds adrenergic GPCRs in skeletal muscle, activating $G_s$. The $G_s\alpha$ subunit stimulates adenylyl cyclase to synthesize $cAMP$, which allosterically activates cyclic-AMP-dependent protein kinase (PKA). Active PKA phosphorylates and activates phosphorylase kinase ($ATP \rightarrow ADP$), which phosphorylates and activates glycogen phosphorylase, triggering rapid glycogen catabolism within seconds.

Page 35: Slow GPCR-cAMP Cascade: Gene Transcription

In slower signaling responses, active PKA translocates through nuclear pores into the nucleus. Nuclear PKA phosphorylates specific transcription regulators, which bind regulatory promoter sequences of target genes to activate transcription. This mechanism regulates hormone synthesis and long-term memory formation over minutes to hours.

Page 36: Cascade Signal Amplification

Signaling cascades provide geometric amplification. A single ligand-receptor binding event activates multiple G proteins and adenylyl cyclases, generating thousands of $cAMP$ molecules that activate kinases, resulting in the generation of $10^8$ molecules of glucose-1-phosphate.

Page 37: Physiological Responses Mediated by cAMP

Hormones stimulate distinct tissues via $cAMP$:

• Epinephrine (Heart): Increases heart rate and force of contraction.

• Epinephrine (Skeletal Muscle): Stimulates glycogen breakdown.

• Epinephrine, Glucagon (Fat): Stimulates fat breakdown (lipolysis).

• Adrenocorticotropic Hormone / ACTH (Adrenal Gland): Stimulates cortisol secretion.

Page 38: Essentiality of Signal Inactivation

Signaling cascades must be rapidly reversible. Permanent locking of a pathway in either an active or inactive state leads to pathology.

Page 39: Cholera Toxin Mechanism and Cystic Fibrosis

Vibrio cholerae secretes cholera toxin, an enterotoxin consisting of subunits A and B that enters intestinal epithelial cells via retrograde endocytosis after binding $GM1$ gangliosides. Subunit A catalyzes irreversible ADP-ribosylation of the $G_s\alpha$ subunit, abolishing its intrinsic $GTPase$ activity.

Locked in a constitutively active state, $G_s\alpha$ drives unregulated adenylyl cyclase activity and massive $cAMP$ production. Continuously elevated $cAMP$ activates the cystic fibrosis transmembrane conductance regulator ($CFTR$) chloride channel, triggering rapid $Cl^-$ efflux, decreased $Na^+$ influx, and massive osmotic water loss ($2\,L/hr$) that leads to severe diarrhea and dehydration.

Heterozygosity for the cystic fibrosis mutation ($CFTR^{+/-}$) provides selective resistance to cholera toxin-induced fluid loss, whereas homozygosity ($CFTR^{-/-}$) causes cystic fibrosis.

Page 40: Enkephalin Regulation of Enterocyte Secretion

Enkephalins counteract cholera-induced fluid loss by binding opioid GPCRs on enterocytes. These receptors engage inhibitory $G_i$ proteins to suppress adenylyl cyclase activity, diminishing $cAMP$ synthesis and halting excessive ion transport.

Page 41: Phospholipase C and Inositol Phospholipid Signaling

Certain GPCRs couple to the $G_q$ protein, whose activated $G_q\alpha$ subunit stimulates the membrane-bound enzyme phospholipase C (PLC). PLC cleaves an inositol phospholipid (phosphatidylinositol 4,5-bisphosphate) located in the inner leaflet of the plasma membrane into two second messengers:

• Diacylglycerol (DAG): Hydrophobic lipid that remains embedded within the plasma membrane.

• Inositol 1,4,5-trisphosphate ($IP_3$): Water-soluble phosphorylated sugar that diffuses freely into the cytosol.

Page 42: Calcium Mobilization and Protein Kinase C Activation

$IP_3$ diffuses to the endoplasmic reticulum (ER) and binds to $IP_3$-gated $Ca^{2+}$ release channels, releasing sequestered $Ca^{2+}$ into the cytosol. Elevated cytosolic $Ca^{2+}$ and membrane-bound DAG together recruit Protein Kinase C (PKC) from the cytosol to the plasma membrane, activating PKC to phosphorylate downstream intracellular proteins.

Page 43: Physiological Responses Mediated by Phospholipase C

Phospholipase C activation regulates various tissue-specific processes:

• Vasopressin (Liver): Induces glycogen breakdown.

• Acetylcholine (Pancreas): Triggers secretion of the digestive enzyme amylase.

• Acetylcholine (Skeletal Muscle): Induces muscle contraction.

• Thrombin (Blood Platelets): Triggers platelet aggregation.

Page 44: Calcium Wave Dynamics at Fertilization

Cytosolic $Ca^{2+}$ spikes serve as ubiquitous biological triggers. Upon sperm entry into an egg cell during fertilization, an explosive wave of released $Ca^{2+}$ sweeps across the cytoplasm, preventing polyspermy and initiating developmental divisions.

Page 45: Calmodulin as an Intracellular Calcium Sensor

Calmodulin is a ubiquitous, highly conserved cytosolic $Ca^{2+}$-binding protein in eukaryotes. It possesses two globular domains connected by a flexible, $2\,nm$ $\alpha$ helix, with each globular domain containing two $Ca^{2+}$-binding sites (four total). Calcium binding triggers a conformational shift that allows calmodulin to wrap around target proteins, such as $Ca^{2+}/\text{calmodulin-dependent}$ protein kinases (CaM-kinases) involved in learning and memory.

Page 46: Functional Scope of Calmodulin Targets

Calmodulin coordinates a vast spectrum of target proteins throughout multiple cellular compartments, including ion channels ($K^+$, $Na^+$, $Ca^{2+}$), receptor tyrosine kinases (EGFR), GPCRs (mGluR7, Angiotensin IIR), cytoskeletal components (myosins, MLCK, caldesmon, spectrin), metabolic enzymes, and transcription factors.

Page 47: Nitric Oxide Signaling in Vasodilation

Blood vessel dilation combines cell-surface and intracellular signaling mechanisms:

1. Acetylcholine binds GPCRs on vascular endothelial cells, activating the $G_q\text{-}IP_3\text{-}Ca^{2+}$ pathway.

2. Increased cytosolic $Ca^{2+}$ stimulates nitric oxide synthase (NOS) to synthesize nitric oxide (NO) gas from arginine.

3. NO rapidly diffuses out of endothelial cells and across the plasma membranes of adjacent smooth muscle cells.

4. Within smooth muscle cells, NO binds to the heme group of soluble guanylyl cyclase, stimulating production of cyclic GMP ($cGMP$) from $GTP$.

5. Elevated $cGMP$ triggers rapid smooth muscle relaxation, expanding vessel diameter and increasing blood flow.

Page 48: Visual Signal Transduction in Rod Photoreceptors

Rod photoreceptor cells utilize a G-protein-mediated cascade adapted for visual signal amplification:

1. In rod outer segments, rhodopsin absorbs a photon, isomerizing 11-cis retinal into all-trans retinal, which dissociates from opsin.

2. Activated opsin stimulates the trimeric G protein transducin ($G_t$).

3. $G_t\alpha$ activates cyclic GMP phosphodiesterase (PDE), which hydrolyzes $cGMP$ into $GMP$.

4. Declining $cGMP$ levels cause cGMP-gated cation ($Na^+$) channels to close, hyperpolarizing the photoreceptor membrane.

5. Hyperpolarization alters neurotransmitter release at the synaptic terminal, disinhibiting postsynaptic retinal neurons to transmit visual signals to the brain.

Amplification scale per photon absorbed:

• $1$ photon activates $500$ transducin molecules.

• $500$ transducins activate $500$ cyclic GMP phosphodiesterases.

• $10^5$ cyclic GMP molecules are hydrolyzed.

• $250$ cation channels close.

• $10^6 - 10^7\,Na^+$ ions per second are blocked from entering for $\sim 1\,s$.

• Membrane potential hyperpolarizes by $1\,mV$.

Page 49: Overview of Enzyme-Coupled Receptors

Enzyme-coupled receptors are transmembrane proteins that display ligand-binding domains on the outer membrane surface and intrinsic catalytic activity (or an enzyme-binding site) on the cytosolic side. The largest family is the Receptor Tyrosine Kinases (RTKs).

Page 50: Activation of Receptor Tyrosine Kinases (RTKs)

RTK monomers span the lipid bilayer as a single $\alpha$ helix. Extracellular ligand binding (often by a dimeric ligand) induces receptor dimerization, bringing cytosolic kinase domains into contact. The adjacent kinase domains trans-autophosphorylate specific tyrosine residues on each other's intracellular tails. Phosphorylated tyrosines serve as high-affinity docking sites for signaling complexes containing SH2 domains, including phospholipase C, Ras-GEF, and PI 3-kinase.

Page 51: Downregulation of RTK Signaling

RTK activity is terminated through two principal mechanisms:

• Dephosphorylation: Protein tyrosine phosphatases (PTPs) remove activating phosphate groups from receptor tyrosines.

• Receptor Endocytosis: Activated receptor complexes are internalized via clathrin-coated vesicles into endosomes and routed to lysosomes for degradation.

Page 52: RTK-Mediated Activation of Ras

Ras is a monomeric GTP-binding switch protein attached to the inner surface of the plasma membrane by a lipid tail. Activated RTKs bind an adaptor protein that recruits a Ras guanine nucleotide exchange factor (Ras-GEF). Ras-GEF stimulates Ras to release $GDP$ and bind $GTP$, shifting Ras into its active state.

Page 53: Ras Molecular Cycling

Ras alternates between an inactive GDP-bound form and an active GTP-bound form. Ras-GEF turns Ras ON by promoting GDP/GTP exchange. Ras-GAP turns Ras OFF by stimulating intrinsic $GTP$ hydrolysis, resetting the switch to transmit further signals.

Page 54: The MAP-Kinase Phosphorylation Cascade

Activated Ras-GTP initiates a three-kinase phosphorelay module known as the mitogen-activated protein (MAP) kinase module:

1. Ras recruits and activates MAP kinase kinase kinase (Raf).

2. MAP kinase kinase kinase phosphorylates and activates MAP kinase kinase (Mek) ($ATP \rightarrow ADP$).

3. MAP kinase kinase phosphorylates and activates MAP kinase (Erk) ($ATP \rightarrow ADP$).

4. MAP kinase phosphorylates downstream effector proteins X and Y (altering protein activity) and transcription regulators A and B (altering gene transcription to drive proliferation).

Page 55: Oncogenic Ras Mutations in Human Cancer

Mutations in Ras oncogenes occur in over $30\%$ of human cancers. Oncogenic mutations inhibit the GTPase activity of Ras, rendering it incapable of hydrolyzing $GTP$ and locking it in a constitutively active Ras*-GTP form that drives uncontrolled cell growth, proliferation, and migration.

Distribution of Ras mutations across human malignancies:

• Pancreatic Cancer: $95\%$ (KRAS)

• Colorectal Cancer: $45\%$ (KRAS)

• Lung Cancer: $35\%$ (KRAS)

• Acute Myeloid Leukemia (AML): $15\%$ (NRAS)

• Melanoma: $15\%$ (NRAS)

• Bladder Cancer: $10\%$ (HRAS)

Page 56: Mapping Phosphorylation Sites by Site-Directed Mutagenesis

Specific tyrosine docking sites on RTKs can be mapped by mutating targeted Tyrosine residues (Tyr, Y; MW $181.2$, $pK_a = 10.46$) to Phenylalanine (Phe, F; MW $165.2$), which lacks the requisite hydroxyl group for phosphorylation. Loss of phosphorylation at mutated sites prevents binding of specific downstream intracellular proteins, identifying which phospho-tyrosine recruits each pathway component.

Page 57: Genetic Ordering of Signaling Cascades

Epistasis experiments using mutant cell lines and constitutively active proteins determine the order of pathway components:

• In cells lacking protein X, introducing overactive Ras restores downstream signaling, proving that Ras acts downstream of protein X.

• In cells lacking protein Y, introducing overactive Ras fails to restore signaling, proving that protein Y acts downstream of Ras.

• Deduced pathway order: $RTK \rightarrow \text{Protein X} \rightarrow Ras \rightarrow \text{Protein Y} \rightarrow \text{Downstream Signaling}$.

Page 58: RTK Activation of PI 3-Kinase

Extracellular survival factors (such as IGF, insulin-like growth factor) bind and activate RTKs, which recruit and activate phosphoinositide 3-kinase (PI 3-kinase). PI 3-kinase phosphorylates inositol phospholipids in the inner plasma membrane leaflet to generate phosphorylated inositol phospholipids ($PIP_3$).

Page 59: Membrane Recruitment and Activation of Akt

Phosphorylated inositol phospholipids provide lipid docking sites on the plasma membrane. Akt (protein kinase B / PKB), a cytosolic serine/threonine kinase, docks at these lipids alongside protein kinase 1. Protein kinase 1 and protein kinase 2 then phosphorylate and activate Akt, allowing it to dissociate and propagate survival signals throughout the cell.

Page 60: Akt-Mediated Cell Survival via Bad Phosphorylation

In the absence of survival signals, unphosphorylated Bad binds to and inactivates Bcl2, an apoptosis-suppressing protein. Activated Akt phosphorylates Bad ($ATP \rightarrow ADP$), causing Bad to release active Bcl2. Free Bcl2 suppresses apoptosis and promotes cell survival.

Page 61: Akt Stimulation of Cell Growth via Tor

Akt promotes cell growth by indirectly activating Tor (target of rapamycin), a large serine/threonine protein kinase. Akt phosphorylates and inhibits an upstream inhibitor of Tor. Activated Tor stimulates protein synthesis and inhibits protein degradation, driving anabolic cell growth and proliferation. The cancer therapeutic rapamycin directly inactivates Tor by binding the FKBP12 complex.

Page 62: Convergence and Cross-Talk Between GPCR and RTK Pathways

Intracellular signaling pathways operate as interconnected networks:

• GPCRs activate adenylyl cyclase (generating PKA) and phospholipase C (generating PKC and CaM-kinase via $Ca^{2+}$).

• RTKs activate phospholipase C (PKC), Ras (MAP kinase), and PI 3-kinase (Akt kinase).

• These five terminal kinases (PKA, CaM-kinase, PKC, MAP kinase, Akt kinase) phosphorylate shared target proteins and transcription regulators to integrate diverse inputs.

Page 63: Notch-Delta Lateral Inhibition

Contact-dependent signaling guides developmental specialization during neurogenesis in Drosophila. A developing neural precursor displays the membrane-bound inhibitory signal protein Delta on its surface. Delta binds the transmembrane receptor Notch on neighboring epithelial cells, activating lateral inhibition to prevent adjacent cells from differentiating into neurons.

Page 64: Direct Proteolytic Route of Notch Signaling

Upon Delta binding, the cytosolic tail of the Notch receptor is cleaved by a membrane-associated protease. The cleaved Notch tail translocates directly into the nucleus, where it binds to DNA-binding complexes and activates transcription of Notch-responsive genes without an intermediate kinase cascade.

Page 65: Nuclear Receptor Superfamily Signaling

Small hydrophobic hormones (cortisol, estradiol, testosterone, thyroxine) diffuse directly across the plasma membrane and bind intracellular receptor proteins residing in the cytosol or nucleus. Hormone binding induces an activating conformational change, allowing the receptor-hormone complex to bind specific regulatory DNA sequences and activate target gene transcription.

Page 66: Ethylene Signaling Pathway in Plants

Plant ethylene receptors are dimeric transmembrane proteins located in the endoplasmic reticulum membrane that act as enzyme-coupled receptors:

• Absence of Ethylene: The unliganded receptor activates an associated protein kinase that directs the continuous degradation of an ethylene-responsive transcription regulator; ethylene-responsive genes remain OFF.

• Presence of Ethylene: Ethylene binds to and inactivates the receptor and its associated kinase, preventing degradation of the transcription regulator. The stabilized regulator translocates to the nucleus and activates the transcription of genes responsible for fruit ripening.

Page 67: Multireceptor Signal Integration

Target cells integrate multiple extracellular inputs through convergent enzymatic phosphorylation. Signal A activates kinase 1, signal D activates kinase 2, and signal B activates both kinases simultaneously, while signal C delivers inhibitory inputs. Full biological activation requires coincidental phosphorylation at both regulatory sites on the target protein, ensuring that complex cellular responses occur only in the presence of the correct combination of cues.