Week 6

• Receptor Tyrosine Kinases (RTKs) have 2 important domains. The N-terminus domain faces the extracellular side of the cell and the C-terminus domain is found facing the cytosol.

  • The N-terminus domain is cysteine rich with many variations
  • the C-terminus domain contains the tyrosine kinase domain, which are very specific.

  
• RTKs auto-phosphorylate on a Tyrosine rich C-terminus

  
• A mitogen is a small peptide or protein that induces a cell to begin cell division. (30-100 AA long)

  
• Mitogenesis is the triggering of mitosis, typically through activation by mitogen

  
• Growth factors are mitogen or mitogen-like molecules that can induce varying effects on the cell including cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription.
• Growth factors can act as mitogens or can alter cell growth, or an essential protein translation process. Note that all GF can be mitogens but not all mitogens are GF.
• in general, many receptor tyrosine kinases (RTKs) are activated by binding to specific growth factors:

  • EGF binds to EGFR and insulin binds to IR (insulin receptor)

  
• Each RTK is expressed in a specific set of cell and tissue types. In general, RTK activation by ligand binding leads to:

  • Proliferation
  • Cell growth
  • Survival
  • Differentiation
  • in some cases, cell migration.
• Some RTKs also control specialized functions in certain cells

  • insulin triggers glucose uptake into muscle and fat cells
  • NGF triggers neuronal migration in brain development

  
• 1 growth factor is usually only going to bind to 1 growth factor receptor
• Tyrosine kinase signalling steps:

  1. Ligand (growth factor) binding to receptor extracellularly causes receptor kinase activation and auto-phosphorylation
  2. Phosphorylated RTKs attract (recruit) specific cytosolic proteins
  3. Some recruited (cytosolic) proteins are phosphorylated, which in turn causes activation.

  
• Tyrosine Kinase signalling:

  1. Ligand (growth factor) binding to the receptor extracellularly causes receptor kinase activation and auto-phosphorylation.
  2. Phosphorylated RTKs attract (recruit) specific cytosolic proteins
  3. some recruited (cytosolic) proteins are phosphorylated, which in turn causes activation

  
• Step 1: ligand binding causes activation of a receptor tyrosine kinase.

  • Inactive RTKs are monomeric.
  • Dimerization occurs during ligand binding
  • It is ON when bonded to ligand (autophosphorylates)
  • Signalling proteins bind to the phosphotyrosine sites
  • these sites are what recruits signalling pathways
  • Each phosphotyrosine could recruit specific adaptor proteins.

  
• Ligand causing receptor dimerization: Some ligands are divalent or can form dimers at specific concentrations. These divalent/dimeric ligands cause receptors to dimerize upon binding.

  
• What if the ligand is a monomer how does it make the receptor dimerize?

  • Ligand binding induces a conformational change in the receptor that causes dimerization.
  • Eg. conformational change causes an area that is hydrophobic --> requires another hydrophobic space to hide the area --> dimerization occurs to keep hydrophobic space away from water.

  
• Recruitment of signalling proteins to phosphorylated receptors:

  • After binding to a ligand, receptor tyrosine kinases are phosphorylated on multiple tyrosine residues
  • Each of these residues acts as a docking site for another protein that is present within the cytosol
  • The "docking proteins" that are recruited to the RTK usually have SH2 or PTB domains that facilitate their interaction

  
• the docking protein for RTK will have a binding site for a specific amino acid side chain and a binding site for phosphotyrosine.

  
• The SH2 domain binds to uniq motifs in proteins ONLY when a key tyrosine residue is phosphorylated (pTyr)

  
• PTB domain like SH2 domains, binds to unique motifs in proteins ONLY when a key tyrosine residue is phosphorylated.

  
• Both SH2 and PTB domains play a large role in the modular nature of signals.

  • most signals activated by receptor tyrosine kinases have SH2 or PTB domain modules

  
• The epidermal growth factor receptor (EGFR) controls various aspects of cell physiology: proliferation, survival, migration, and metabolism.

  
• • EGF - Epidermal growth factor, stimulates cell growth and proliferation
  • EGFR - RTK that is activated when it binds to EGF
  • Grb2 - growth factor receptor bound protein 2, adaptor protein containing an SH2 domain (recruits Sos)
  • Sos - son of sevenless; it is a Ras Gef
  • Ras - GTPase for the MAPk cascade
  • Raf - Ser/Thr protein kinase
  • Mek - Tyr/Thr protein kinase
  • Erk - Ser/Thr protein kinase.

  
• EGF is a monomeric ligand. It activates the EGFR by inducing a conformation change that results in dimerization of the receptor.

  
• • Ligand binding to the receptor causes the receptor to change conformation, dimerize, and autophosphorylate
  • Phosphorylated receptor leads to binding of the protein Grb2 (contains SH2 domain), which recruits SOS (Ras GEF)
  • Ras is loaded up with GTP by SOS
  • GTP-bound Ras is active, leading to downstream signalling of the MAPK cascade starting with Raf
  • activated Raf phosphorylates Mek
  • Mek phosphorylates Erk
  • Erk phosphorylates many cellular targets

  
• Many cancers have a mutation in Ras that enhances Ras function.

  • Increase Ras activation with lower fidelity of growth factor, increases the activity of cell whether it's through growth, division, or motility.

  
• Once activated, Erk activates specific transcription factors and other cellular pathways, which all lead to enhanced PROLIFERATION and GROWTH.
• Phosphatidylinositol is a glycerophospholipid with inositol as a headgroup. Inositol headgroup can be phosphorylated on three different positions by specific lipid kinases in response to specific cues.

  
• Phosphorylated phosphatidylinositols are called phosphoinositide phosphates (PIPs). PIP2 is abundant in the plasma membrane.

  
• PI3K-Akt-mTOR pathway:

  • GAB - adaptor protein (Grb2 associated binding protein)
  • PI3K - phosphoinositide-3-kinase phosphorylates PIP2
  • PIP3 - recruits and localize PDK1 and Akt
  • PKD1 - ser/Thr kinase that acts on Akt
  • Akt - a ser/Thr Kinase that acts on many targets. To be activated it has to be turned on by mTOR (complex 2) and PDK1
  • mTOR - ser/Thr kinase that acts on Akt
  • PTEN - phosphatase that acts on PIP3 --> turning it back into PIP2.

  
• PI3K-Akt-mTOR pathway

  • EGF activates EGFR --> which autophosphorylates using ATP
  • GAB then attaches to the tyrosine phosphate and becomes activated. Activated GAB recruits P13K to RTK
  • phosphorylated PI3K is activated by RTK to phosphorylate PIP2 into PIP3
  • PIP3 is an anchor molecule PDK1 and Akt.
  • PDK1 is activated when it binds to PIP3
  • to activate Akt it requires phosphorylation of PDK1 and mTOR (complex 2).
  • Akt then detaches from PIP3 and performs kinase activities in different pathways