Tyrosine kinases

• Tyrosine kinase activation:

  • Signal protein binds to extracellular side of the receptor
  • Causes conformational change to intracellular domains
  • Intracellular domains are autophosphorylated, activating kinase domains
  • Autophosphorylation generates binding sites for signalling proteins
• There are 58 tyrosine kinases encoded in the human genome
• Four examples of RTKs and their corresponding signal protein family:

  • EGF receptors (epidermal growth factors) - stimulates cell survival, growth and proliferation
  • Insulin receptors (insulin) - stimulates carbohydrate utilisation and protein synthesis
  • Trk receptors (nerve growth factors) - stimulates survival and growth of some neurons
  • FGF receptors (fibroblast growth factor) - stimulates proliferation of various cell types and inhibits proliferation of some precursor cells
• Receptor tyrosine kinases are frequently over-expressed or mutated in cancer
• Anti-phosphotyrosine Western blots are used to study receptor tyrosine kinase activation:

  • Ligand suspected to activated RTK is incubated with RTK.
  • Western blot with anti-pTyr - if the RTK is activated the tyrosine residues in the binding sites will be autophosphorylated so anti-pTyr will bind and show up on the Western blot.
• Western blotting:

  • Extract proteins from cells by lysing them e.g. via sonication then homogenise then centrifuge. Add sample buffer
  • Carry out SDS-PAGE. Blot the gel onto a membrane
  • Incubate in blocking solution to prevent nonspecific binding of antibodies
  • Incubate with primary antibody that will bind to the target protein - in the case of RTKs use anti-pTyr
  • Wash away any excess primary antibody. Incubate with secondary antibody that is linked to a tag e.g. GFP.
  • The secondary antibody will bind to the primary antibody
  • Interpret results for different combinations of ligand + receptor
• Most RTKs are dimeric so having two domains on the ligand allows one ligand domain to bind per extracellular domain pulling the intracellular RTK domains together to dimerise
• Give an example of a monomeric RTK ligand and describe the mechanism of how it dimerises the RTK:

  • EGF = epidermal growth factor
  • Binds to monomeric EGF receptor (i.e. extracellular portion) causes dimerisation arm to be exposed which promotes EGFR dimerisation
• Describe how the insulin receptor is dimerised:

  • Extracellular region is covalently linked by disulfide bonds but intracellular domains are not so are inactive
  • Binding of insulin to extracellular region causes dimerisation of intracellular domains
  • So the receptor is still made up of two domains but they are only covalently linked extracellularly - this is different to other RTKs which require dimerisation of both the extracellular and intracellular regions to allow for receptor activation
• How RTKs are activated in terms of the tyrosine residues:

  • In its non-phosphorylated state (i.e. in the absence of effector binding), the activation loop prohibits substrate binding and holds Tyr1162 in the binding site as a pseudosubstrate
  • Extracellular binding of effector molecule causes autophosphorylation of tyrosine residues in the activation loop (Tyr1158 Tyr1162 and Tyr1163).
  • Activation loop moves out of the binding site accomodates the Tyr residue of a substrate - downstream signalling mechanism
• The downstream signalling molecule activated by a receptor tyrosine kinase is typically a tyrosine kinase that is not bound to the lipid membrane i.e. is not a receptor - phosphorylation of the Tyr residues in the activation loop of the subtrate tyrosine kinase causes a downstream tyrosine kinase cascade
• What is the term for when one tyrosine kinase phosphorylates another tyrosine kinase? Transphosphorylation
• The EGF receptor tyrosine kinase is activated by an allosteric mechanism:

  • Binding of EGF to extracellular binding site causes dimerisation of receiver and activator domains
  • Activation caused by dimerisation causes the autophosphorylation of the C-terminal tails of the domains
• Three examples of intracellular signalling proteins that dock to phosphorylated tyrosine residues in the RTK active site:

  • Phospholipase C-gamma: leads to activation of protein kinase C and Ca2+ release from ER stores
  • Phosophoinositide 3-kinase: phosphorylates lipid head groups which become docking sites for effectors
  • Non-enzymatic adaptors (Grb2): leads to activation of small GTPase Ras and downstream signalling (MAP kinase pathway)
• The SH2 domain recognises phosphotyrosines to permit specific binding of the downstream signalling proteins to the tyrosine kinase. It does this as there is an apolar specificity pocket for the residue in the +3 position i.e. 3 amino acids away from the pTyr
• Mitogen-activated protein kinases are only activated when the cell is undergoing mitosis
• The Ras-MAP pathway is central to the regulation of cell growth and proliferation; it is deregulated in many types of cancer
• Ras-MAP kinase pathway from activation of RTK to influencing the cell cycle:

  • RTK activated
  • Grb2
  • Sos
  • Ras
  • Raf
  • MAPKK
  • MAPK
  • Cell cycle
• Ras is a small GTPase
• Post-translational modifications anchor Ras to the cell membrane: 2 Cys residues bound to lipid anchors form disulfide bridges with Ras
• How signal amplification specificity and regulation is achieved in the Ras-MAP kinase signalling pathway:

  • SH2 domain of Grb2 binds to pTyr on activated RTK
  • Conformational change in SH3 domain; activates Sos (Ras-GEF)
  • Sos exchanges GDP for GTP in the Ras binding site; Ras is localised nearby as it is bound to the inner leaflet of the lipid membrane
  • Active Ras protein performs downstream signalling
• How does Ras-GTP activate the downstream MAP kinase pathway? Via allosteric activation - firstly of Raf PK which phosphorylates Mek PK which phosphorylates Erk PK in a kinase cascade
• The two outcomes of the MAP kinase pathway:

  • Phosphorylation of protein which changes protein activity
  • Phosphorylation of transcription factors which changes gene expression
• Scaffolding proteins ensure specificity of the MAPK pathway. They assemble the specific protein kinases sequentially to ensure this pathway is followed specific to the effector molecule that binds extracellularly
• Tyrosine kinase inhibitors are synthesised specific to the problematic receptor tyrosine kinase posing a cancer risk. Binds to extracellular receptor domain but does not induce conformational change/activation of intracellular catalytic domains. Monoclonal antibodies can also interfere with receptor tyrosine kinases
• Apart from the p-Tyr Western blot, receptor tyrosine kinase activation can be studied by:

  • Monitoring cellular responses: by quantifying DNA synthesis to monitor proliferation or by direct microscopic observation of cellular behaviour.
  • Looking out for clustering rapid mitosis, cytoplasmic growth, morphological changes etc.
• Scaffolding proteins are necessary to ensure downstream signalling specificity as there are many different types of Raf MEK and Erk protein kinases. Therefore, there are many combinations - specificity necessary to achieve desired result i.e. specific protein modification or change to gene expression
• How Ras is activated:

  • Guanine exchange factor (GEF) swaps GDP for GTP in the Ras binding site
  • This activates Ras as the switch helix interacts with Ras effectors
  • When GAP cleaves the gamma-phosphate from the GTP to convert it to GDP the switch helix in Ras moves and no longer interacts with the Ras effectors