Signal Transduction 3

Liver Glycogen and Glucose Regulation

• Glycogen Breakdown to Glucose

  • Glycogen in the liver is broken down to glucose.

  • Hormonal regulation by glucagon and insulin.

  • Glucagon: Promotes glucose release from the liver.

  • Insulin: Inhibits glucose release and enhances glucose uptake by cells.

  • Regulatory Mechanism:

  • They antagonistically regulate glucose release in the liver through the action of cyclic AMP (cAMP).

  • Glucagon triggers cAMP production, enhancing glucose release.

  • Insulin decreases cAMP levels, thus reducing glucose release.

  • Role of GLP-1 Agonists:

  • Semaglutide and tirzepatide are glucagon-like peptide 1 (GLP-1) agonists.

  • Function: Attenuate glucagon production and boost insulin release in a glucose-dependent manner.

Receptor Tyrosine Kinases (RTK)

• Prevalence: Approximately 50 receptor tyrosine kinase (RTK) types identified in the human genome.

• Functions:

  • Involved in processes such as cell proliferation, differentiation, and migration.

  • Play critical roles in development and tissue homeostasis.

• Insulin Receptor:

  • A member of the tyrosine kinase receptor superfamily.

Characteristics of RTKs

• Structural Features:

  • Composed of transmembrane proteins.

  • Catalytic receptors with ligand binding sites located outside of the cell.

  • Intracellular region has a protein kinase active site.

• Kinase Specifications:

  • Specifically phosphorylates tyrosine residues.

  • Activation of the protein kinase upon ligand binding leads to autophosphorylation (self-phosphorylation) of tyrosine residues within the intracellular section of the receptor.

Mechanism of Tyrosine Kinase Receptor Activation

• Autophosphorylation process:

  1. Dimerization:

  • Ligand binding typically causes the receptors to dimerize (pair up).

  1. Activation:

  • Dimerization changes the conformation of catalytic domains from inactive to active, although they can revert to inactive state due to inhibition from the activation loop.

  1. Cross-Phosphorylation:

  • Dimerization allows cross-phosphorylation, activating the receptors.

  1. Phosphorylation of Intracellular Residues:

  • Results in phosphorylation of additional intracellular tyrosine residues.

  1. Ligand-induced conformational changes:

  • Physical changes lead to subsequent phosphorylation events.

• Types of Dimerization:

  1. Ligand-dependent (TRKA)

  2. Receptor-dependent, but not through ligand interaction (EGF)

  3. Bi-ligand interaction through receptor interface (KIT)

  4. Involvement of ligand, receptor, and accessory proteins (FGFR with heparan sulfate).

Activation Loop in Kinase Functionality

• Inactive Kinase:

  • The activation loop occludes the active site and prevents substrate binding.

• Activation Process:

  • Tyrosine phosphorylation at specific residues (e.g., T1162 and T1163) disrupts existing intramolecular interactions, allowing the activation loop to reposition and liberate the active site.

  • This facilitates coordination with ATP and enables substrate access for kinase activity.

• Significance:

  • The stabilization of the active conformation of insulin receptor tyrosine kinase (IRK) is critical for its functions.

SH2 and PTB Domains in Signaling

• SH2 Domains:

  • Present in about 100 proteins primarily interacting with phosphotyrosine residues.

  • Approximately 100 amino acids in size, forming a compact structure with beta-sheets and binding pockets.

• PTB Domains:

  • 27 PTB domain containing proteins are found in the human genome and primarily interact with non-phosphorylated tyrosine residues.

  • Example: 1 SH2 domain in yeast indicates low levels of tyrosine kinase signaling there.

• Signaling Functionality:

  • Different SH2 domains show specificity by interacting with distinct tyrosine phosphorylated peptides, allowing diverse outputs from tyrosine kinase signaling.

Downstream Signaling Through Direct Phosphorylation

• PI3K Activation:

  1. The regulatory subunit (P85) of phosphoinositide 3-kinase (PI3K) interacts with phosphorylated receptors, becoming active and producing phosphatidylinositol (3,4,5)-trisphosphate (PIP3).

  2. Phospholipase C gamma (PLCγ) is also recruited and activated via its SH2 domain, leading to the production of diacylglycerol (DAG) and inositol trisphosphate (IP3).

  3. Grb2, through its SH2 domain, helps activate the RAS/MAPK pathway.

• Regulatory Implications:

  • Tyrosine phosphorylation of receptors drives interactions with and regulation of downstream molecules, influencing various cellular responses.

Recruitment of Adaptor Proteins in Signaling

• Mechanism:

  • Adaptors engage with the receptor via phospho-tyrosine binding, leading to multiplication of phosphorylation events and initiating downstream signaling.

  • Example Adaptors:

  • GAB1 (EGF and other receptors)

  • IRS1 (Insulin and IGF-1 receptors)

Internalization Pathways of RTKs

• Ubiquitination:

  • Tyrosine phosphorylation induces ubiquitination via Cbl protein, promoting internalization of receptors.

• Pathways:

  • Coupling reader modules for signaling (e.g., ubiquitylated receptors enter endocytic pathways leading to lysosomal degradation or recycling).

RTK Signaling Overview

• Pathways Regulated by RTKs:

  • PI3K

  • PLC

  • RAS

  • These pathways affect cell migration, proliferation, and metabolism.

• Phosphatidylinositol synthesis:

  • Functional lipid second messenger, critical for understanding the action of RTKs and GPCRs in intracellular signaling.

Key Enzymes and Their Functions

• Key Enzymes:

  • PI3K: Converts PtdIns(4,5)P2 to PtdIns(3,4,5)P3.

  • PTEN: A tumor suppressor removing phosphate, regulating PtdIns(3,4,5)P3 levels.

  • SHIP1/2: 5-phosphatases generating PtdIns(3,4)P2 from PtdIns(3,4,5)P3.

• Pathway Regulation:

  • Balance of enzymatic activity controls PtdIns(3,4,5)P3 generation kinetics and impact on signaling outputs.

Mechanistic Regulation by PI3K Activity

• Class 1A PI3K Activation:

  • p110 catalytic subunits and p85 regulatory subunits regulate enzyme activity.

• Activation Steps:

  1. SH2 domain of p85 engages with YxxM motifs on phosphorylated receptors or adaptors.

  2. This action relocates and activates PI3K at the membrane where it encounters its substrate, PtdIns(4,5)P2.

Reader Output Mechanism from PtdIns(3,4,5)P3

• Signal Cascades:

  • PtdIns(3,4,5)P3 interacts with proteins containing specific motifs (e.g., Pleckstrin homology (PH) domain).

  • Recruitment of downstream signaling players is initiated by binding of PtdIns(3,4,5)P3.

Insulin Signaling Pathway

• Insulin Receptor Activation:

  • PI3K is activated through receptor interaction, generating PIP3, with downstream effects including:

  • Glucose uptake via Glut4 translocation.

  • Glycogen accumulation through GSK inactivation.

  • Insulin also enhances lipid and protein synthesis by modifying several pathways via AKT/PKB signaling, influencing cellular metabolism significantly.

RAS Signaling Pathways

• Small GTPases:

  • RAS family includes RAS, Rac, Rho, CDC42, ARF, RAB.

  • Function as molecular switches controlling various cellular effects; >150 members identified.

  • Include several regulatory proteins which impact phosphoinositide pathways (PH motifs present in many).

• GTPase Cycle:

  • RAS undergoes conformational changes upon GTP hydrolysis, mediating downstream protein interactions and cellular responses.

Mutational Implications and Oncogenesis

• Mutations in RAS:

  • Common mutations include G12, G13, and Q61 which maintain RAS in an active state, contributing to proliferation and oncogenesis.

  • The role of RAS in cancer is evidenced by prevalence in pancreatic tumors and its correlation with various tissue types (e.g. K-RAS in colon cancer).

Growth Factors and Their Functions

• Classes of Factors:

  • PDGF: Promotes connective tissue proliferation (dimer forms: AA, AB, BB).

  • EGF: Stimulates proliferation of mesenchymal, glial, and epithelial cells.

  • TGF-α: Found in transformed cells, may play roles in wound healing.

  • FGF: Involves broad cell proliferation activities.

  • NGF: Critical for neural cell survival and neurite outgrowth.

  • IGF-I/II: Promote numerous cellular proliferative activities.

SH2 Domain Proteins and Their Regulatory Role

• Functional Overview:

  • SH2 domain proteins are pivotal in regulation and signaling; their diversity dictates the specificity of RTK signaling pathways.

• Mutation Influence:

  • Mutations in PTEN and PI3K frequently link to tumorigenesis, emphasizing the significance of signaling stability in maintaining cellular homeostasis.

Liver Glycogen and Glucose Regulation

• Glycogenolysis in the Liver

  • Glycogen in the liver acts as a critical glucose reservoir to maintain blood sugar levels during fasting. The process is catalyzed by glycogen phosphorylase and regulated by the enzyme glucose-6-phosphatase, which allows glucose to be released into the bloodstream.

  • Hormonal Regulation:

  • Glucagon: Secreted by alpha cells of the pancreas. It binds to G-protein coupled receptors (GPCRs), stimulating adenylate cyclase to produce cyclic AMP (cAMP). High levels of cAMP activate Protein Kinase A (PKA), which triggers a phosphorylation cascade ending in the activation of glycogen phosphorylase.

  • Insulin: Secreted by beta cells of the pancreas. It acts via the Insulin Receptor (an RTK) to activate phosphodiesterases that degrade cAMP, effectively counteracting glucagon and promoting glycogen synthesis (glycogenesis) via Glycogen Synthase.

  • GLP-1 Agonists (Incretin Mimetics):

  • Compounds like Semaglutide and Tirzepatide mimic the action of Glucagon-like Peptide-1.

  • They enhance insulin secretion and suppress glucagon release in a glucose-dependent manner, meaning they primarily function when blood glucose is elevated, reducing the risk of hypoglycemia.

Receptor Tyrosine Kinases (RTK) Architecture

• Prevalence and Diversity:

  • There are approximately 50 to 58 known RTKs in humans, categorized into 20 subfamilies.

  • They regulate fundamental cellular processes: cell cycle progression (proliferation), specialized cell functions (differentiation), and cell movement (migration).

• Insulin Receptor (IR):

  • Unique among RTKs, the insulin receptor (and IGF-1 receptor) exists as a pre-formed disulfide-linked heterotetramer (̑2̒2). Ligand binding induces a conformational change rather than simple dimerization of monomeric subunits.

Characteristics and Structural Features of RTKs

• Domain Organization:

  • Extracellular Domain: Contains the ligand-binding site, often enriched with leucine-rich repeats or cysteine-rich clusters.

  • Transmembrane Helix: A single hydrophobic alpha-helix anchoring the receptor in the lipid bilayer.

  • Intracellular Region: Includes a juxtamembrane regulatory segment, the tyrosine kinase catalytic domain, and a C-terminal tail containing multiple tyrosine residues.

• Kinase Activity:

  • The kinase is specific for the phenol group of tyrosine. Activation leads to autophosphorylation, where the receptor phosphorylates its own cytoplasmic tail, creating docking sites for downstream signaling proteins.

Mechanism of Tyrosine Kinase Receptor Activation

• The Activation Process:

  1. Ligand Binding and Dimerization: Except for the Insulin Receptor, most RTKs (like EGFR or PDGFR) are monomeric and require ligand binding to induce dimerization.

  2. Trans-phosphorylation: Once dimerized, the kinase domain of one receptor subunit phosphorylates the tyrosine residues on the opposing subunit (cross-phosphorylation).

  3. Conformational Change: Phosphorylation of the activation loop (A-loop) moves it out of the catalytic site, allowing ATP and substrates to bind.

• Dimerization Models:

  • Ligand-mediated: The ligand is multivalent and bridges two receptors (e.g., PDGF).

  • Receptor-mediated: Ligand binding exposes a dimerization interface on the receptor itself (e.g., EGF).

The Activation Loop and Molecular Switches

• Structural Inhibition:

  • In the inactive state, the activation loop sits in the active site, physically blocking the entry of ATP and protein substrates.

• Molecular Trigger:

  • In the Insulin Receptor Kinase (IRK), phosphorylation of residues T1162 and T1163 (within the A-loop) causes the loop to swing out. This stabilizes the "active" conformation and provides the structural coordination required for the catalytic transfer of phosphate from ATP to the target protein.

SH2 and PTB Domains: Modular Signaling

• SH2 (Src Homology 2) Domains:

  • Composed of ~100 amino acids, these domains recognize a phosphorylated tyrosine (pY) followed by a specific three-amino-acid sequence (motif: pY-X-X-Z). This specificity allows distinct RTKs to recruit specific sets of signaling molecules.

• PTB (Phosphotyrosine-Binding) Domains:

  • These recognize an NPXY motif (asparagine-proline-any-phosphotyrosine). While some PTB domains require phosphorylation, others can bind non-phosphorylated motifs.

• Example: The presence of only 1 SH2 domain in the yeast genome (S. cerevisiae) underscores that RTK signaling is a complexity primarily associated with multicellular metazoans.

Major Downstream Signaling Cascades

• PI3K/AKT Pathway:

  1. The p85 regulatory subunit of PI3K binds to pY residues on the receptor or adaptor proteins (like IRS-1).

  2. This activates the p110 catalytic subunit, which converts PI(4,5)P2 into PI(3,4,5)P3 (PIP3) at the plasma membrane.

  3. PIP3 acts as a docking site for proteins with Pleckstrin Homology (PH) domains, such as AKT (PKB) and PDK1.

• PLC̑ Pathway:

  • Recruitment of Phospholipase C gamma via its SH2 domain leads to the cleavage of PIP2 into DAG (activating Protein Kinase C) and IP3 (releasing intracellular Ca^{2+}).

• RAS/MAPK Pathway:

  • The adaptor protein Grb2 binds the receptor and recruits Sos (a Guanine Nucleotide Exchange Factor or GEF). Sos activates RAS by exchanging GDP for GTP.

Internalization and Deactivation

• Ubiquitination: The E3 ubiquitin ligase Cbl binds to phosphorylated RTKs via its SH2 domain. This tags the receptor with ubiquitin, signaling for its internalization via clathrin-coated pits.

• Fate of the Receptor: Once internalized, the receptor is either sorted to the lysosome for degradation (terminating the signal) or recycled back to the cell surface to sustain sensitivity.

Key Enzymes and Tumor Suppressors

• PTEN (Phosphatase and Tensin Homolog):

  • A critical tumor suppressor that dephosphorylates PIP3 back to PIP2, effectively "turning off" the survival signal from the PI3K/AKT pathway.

• SHIP1/2:

  • 5-phosphatases that convert PIP3 to PIP(3,4)2, modulating the duration and intensity of the lipid signal.

RAS Small GTPases and Cancer

• The GTPase Cycle:

  • RAS acts as a binary switch. It is Active when bound to GTP and Inactive when bound to GDP. GAPs (GTPase Activating Proteins) accelerate the intrinsic GTP hydrolysis of RAS to turn it off.

• Oncogenic Mutations:

  • Mutations at codons G12, G13, or Q61 are the most common. These mutations inhibit the ability of GAPs to stimulate GTP hydrolysis, locking RAS in the "ON" state. This leads to constitutive signaling for cell growth, a hallmark of cancers like pancreatic and colorectal adenocarcinoma.