Principles of Signal Transduction - Lecture Notes

HUBS2206 Human Biochemistry and Cell Biology Lecture 26: Principles of Signal Transduction

Learning Targets

  • Signal Transduction Cascade: Understanding what it is and how it works.
  • Extracellular Receptors: Identifying different types.
  • Protein Kinases and Phosphatases: Understanding their roles, specifically Tyr and Ser/Thr (de)phosphorylation of proteins.
  • Phosphorylation Cascade: Understanding and knowing the MAP kinase cascade.
  • Death Receptor Signalling: Understanding its role in apoptosis.
  • Signal Termination: Understanding how and why it is important.
  • Key Concept: Altered signalling cascades are associated with miscommunication and disease.

Signal Transduction via Cell-Surface Receptors

  • A signal molecule binds to a receptor protein, which activates intracellular signal molecules, ultimately altering target proteins to create a response.

Cell-Surface Receptors

  • Most signalling molecules are hydrophilic and cannot enter the cell, thus they act through cell-surface receptors.

Cellular Response to External Signals

  • Cells respond differently to the same external signal.
  • Cells must possess a receptor for a particular signalling molecule to respond.
  • The actual response depends on the intracellular machinery that integrates and interprets the signal.
  • The same signalling molecule can elicit different responses in different cells (e.g., acetylcholine).

Overview: Flow of Information

  • Hormone/Ligand: Acts as the first messenger.
  • Receptor: Responsible for the reception of the signal.
  • Signal Transduction: Involves second messengers, relay, and signalling molecules.
  • Response: Involves changes in effectors.

Cell-Surface Receptors: Types

  • Enzyme-linked receptors: (see lecture 27)
  • G-protein coupled receptors (GPCRs): (see lecture 28)
  • Ion channel-coupled receptors: (see lecture 29)

Complexity of Signal Transduction Pathways

  • Extracellular ligand binding to a receptor is converted into complex intracellular signals.
  • The pathway involves primary transduction, relay, amplification, integration, spreading, anchoring, and modulation within the cytosol and nucleus.
  • These processes lead to activated gene transcription and effector protein activation.

Complexity of Signal Integration

  • Cells integrate multiple signals through multiple cell-surface receptors.
  • Signals can be integrated in different ways:
    • One receptor activates multiple pathways.
    • Different receptors activate the same pathway.
    • Different receptors activate different pathways, where one pathway affects the other.

Failure of Cellular Communication

  • Signalling is hijacked in diseases like cancer.
  • Examples:
    • Motility circuits: involving proteases, E-cadherin, and integrins.
    • Proliferation circuits: involving growth factors, tyrosine kinases, Ras, Myc, hormones, and cytokines.
    • Cytostasis and differentiation circuits: involving anti-growth factors, p21, p53, and Smads.
    • Viability circuits: involving DNA-damage sensors, abnormality sensors, and death factors.

Importance of Studying Cellular Signalling

  • Understanding molecular mechanisms of disease.
  • Comparative study of signalling pathways in “normal” versus diseased cells.
  • Most cancer-associated modifications of cell signalling are yet to be fully elucidated.

Therapeutic Strategies from Understanding Cell Signalling

  • Signalling understanding aids development of new therapeutic strategies.
  • Many current drugs target ligands, receptors, and key signal transduction molecules.
  • Examples:
    • Antibodies as drugs to bind ligands or receptors, preventing receptor activation.
    • Drugs mimicking ligands to enhance signalling.
    • Drugs inhibiting protein kinase activity.

Critical Role of Protein Phosphorylation in Signal Transduction

Regulation of Protein by Phosphorylation

  • Approximately 1/3 of proteins are regulated by phosphorylation/dephosphorylation, primarily on Ser, Thr, or Tyr amino acids.
  • Mediated by protein kinases and phosphatases.
  • An active protein kinase transfers a phosphate group from ATP onto a protein substrate.
  • An active protein phosphatase dephosphorylates the protein (removes the phosphate group).

Role of Protein Phosphorylation

  • Changes in phosphorylation state of the substrate are associated with protein conformational (shape) changes.
  • Changes in phosphorylation state can alter:
    • Protein activity (phosphorylation often leads to activation, but the reverse can occur).
    • Protein interactions (phosphorylation can promote or detach protein binding).
    • Distribution within the cell (e.g., translocation from cytosol to nucleus or plasma membrane).
  • Many other post-translational modifications (PTMs) such as acylation, methylation, glycosylation, and ubiquitination regulate activity, levels, and/or distribution of proteins.

Protein Kinases and Phosphatases: Numbers and Regulation

  • In Homo sapiens, among ~23,000 genes, ~2-4% encode kinases or phosphatases.
  • Substrates of kinases or phosphatases can be:
    • Other kinases or phosphatases.
    • Receptors.
    • Metabolic enzymes.
    • Cytoskeletal, scaffolding, and nuclear proteins and transcription factors.
    • Ion channels.
  • Many factors regulate the activity of protein kinases and phosphatases:
    • Binding of activators/inhibitors (proteins, lipids).
    • Ions (e.g., calcium, magnesium).
    • Signalling molecules and second messengers (e.g., cAMP).
    • Phosphorylation and other post-translational modifications.
  • Approximately ~200 protein phosphatases (Tyr and Ser/Thr phosphatases).
  • Approximately ~518 protein kinases (90 Tyr kinases and 428 Ser/Thr kinases).

Signalling by Phosphorylation

  • ON switch: Typically kinase-mediated protein phosphorylation.
  • OFF switch: Typically phosphatase-mediated dephosphorylation.
  • However, there are exceptions where phosphorylation turns off signals, and dephosphorylation turns them on.

Typical Phosphorylation Cascade

  • In quiescent cells, many protein kinases are in an inactivated state.
  • Upon cell stimulation, they become phosphorylated, resulting in their activation.
  • Activation of signalling cascades leads to an altered balance between kinase and phosphatase activity.

Typical Phosphorylation Cascade: MAP Kinase Cascade

  • Mitogen-activated protein kinases (MAPK or ERK) integrate various extracellular signals.
  • They target cytoplasmic and nuclear (transcription factors) proteins.
  • The MAPK cascade is a series of 3 protein kinases:
    • Receptor activation leads to activated Raf.
    • Activated Raf phosphorylates and activates MEK.
    • MEK phosphorylates and activates ERK.
    • ERK phosphorylates other proteins.
    • Results in activation of pre-existing proteins and changes in gene expression.
    • Important for the control of cell growth and survival.

Organisation of Signalling Pathways

  • The specific and appropriate response of cells to external stimuli requires integration of multiple signalling pathways.
  • Stimulation of cell surface receptors initiates cellular signals governed by post-translational modifications (e.g., phosphorylation).
  • To increase specificity, the way information is transferred inside the cell is highly organised:
    • Proteins may need recruitment to specific subcellular locations like plasma membrane microdomains.
    • Adaptor proteins: small; contain protein-binding modules that link 2 proteins together, facilitating the creation of larger signalling complexes.
    • Protein scaffolds or anchor proteins help relay the message by serving as a docking site for multiple signalling proteins involved in the pathway.
    • They can also regulate the activity of proteins in these multi-protein complexes.
    • Docking proteins are similar but localize at the membrane next to an activating receptor, to which they bind in a phosphorylation-dependent manner.

Spatial Organisation of Signalling Pathways

  • Information is often transferred in a highly organized manner.
  • Signalling components are proteins, information is transmitted through protein-protein interactions using signal transduction domains.
  • Scaffolds function to hold together individual components of signalling pathways to create macromolecular signalling complexes.
  • These complexes can aggregate in specific locations within the cell, as occurs in lipid rafts and caveolae.

Signalling by Death Receptors

  • Death receptors are members of the tumour necrosis factor receptor superfamily that can mediate caspase activation and apoptosis.
  • Assembly of DISC (Death-inducing signalling complex):
    • TNF ligand binds as a trimer and activates transmembrane receptor (trimer).
    • Recruitment of adaptor proteins (TRADD, FADD) that interact with ‘death domains’ present in the receptor (cytoplasmic side).
    • Adaptor proteins recruit additional pathway-specific enzymes to the TNF-R1 complex: Formation of DISC leads to activation of caspases and apoptosis.

Termination of Signalling

Turning Off the Signal

  • Multiple ways to turn off the signal at several levels:
    1. Removal of Ligand:
      • Most ligands rapidly fall off the receptor.
      • Most ligands are short-lived and rapidly removed from circulation or degraded in the extracellular space (e.g., half-life of circulating peptide hormones is approximately a few minutes).
    2. Receptor Level:
      • Inactivation by dephosphorylation or binding of inhibitory protein.
      • Internalisation leading to receptor degradation (through lysosomal digestion), recycling, or sequestration.
      • Desensitisation: the receptor no longer responds to the signal (e.g., insulin resistance).
    3. Intracellular Signal Transduction Molecules:
      • Inactivation (often via dephosphorylation by protein phosphatases or binding of an inhibitory protein).
      • Degradation/removal.
      • Changes in localisation or sequestration.
  • Turning the signal “off” is critical to restore the inactive state for homeostasis.
  • Persistent activation of growth factor signalling leads to cancer.

Summary

  • Signal transduction can occur via activation of cell-surface receptors.
  • Activation of receptors leads to a signal transduction cascade that relays the message via signalling molecules.
  • This results in a change in effectors to induce a cellular response.
  • Signal cascades are highly complex, and multiple cascades/pathways often intersect (cross-talk).
  • Dysregulation of signalling cascades leads to disease.
  • Phosphorylation/dephosphorylation is a major mechanism of intracellular signal transduction.
  • Termination of the signal is just as important as initiation.