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.