L2 - Action of cytokines + signalling cascades

• What does the cellular response of a cytokine depend on?

  The cell type despite the use of the same signalling pathway
• What pathway is induced when EPO is recognised?

  JAK/STAT pathway
• Is EPO recognised extracellularly or intracellularly?

  Extracellularly - on the outside of the cell
• What is the JAK/STAT pathway important for?

  Differentiation, regulation and cell development
• What is the structure of EPO, EPO-receptor and JAK?

  EPO (ligand) binds to the ligand binding site which is the EPOR (receptor) - all extracellular

  Transmembrane alpha helix in the cytosol - JAK kinase is associated with the receptor
• What happens when EPO binds to its receptor?

  Ligand binds to the binding site of 2 receptors - brings them together (dimerisation)

  Dimerisation brings the JAK kinases close together - they phosphorylate and activate each other

  The signal crosses the membrane but EPO does not
• What happens after the JAK kinases are activated?

  They hyperphosphorylate the tail residues of the receptor (below the kinases)

  SH2 domain of STAT transcription factor (multidomain protein) binds and recognises the phosphorylated tail residues

  JAK phosphorylates STAT and it is released and dimerises with another phosphorylated STAT - translocates to the nucleus and activates transcription
• How do phosphorylated STAT molecules now to translocate to the nucleus?

  When they dimerise, the NLS is exposed - nuclear localisation sequence

  The STAT dimer is now localised to the nucleus
• How does the SH2 domain bind to the phosphorylated residues on the EPOR?

  Tyrosine residue on the SH2 domain is phosphorylated by JAK - acts as an anchor

  Anchor + peptide sequence - binding of the SH2 domain to the phosphorylated tail residues
• How does STAT bind DNA?

  Has positively charged binding domains in the middle of its structure - binds negatively charged DNA
• What part of STAT gets phosphorylated by JAK?

  C-terminus - SH2 domain
• How does STAT dimerise?

  Phosphate tail of one monomer binds to SH2 domain of the other monomer - cross link structure
• How are the actions of cytokines regulated and turned off?

  Short term adaptation - quick and can outcompete a part of the cascade

  Long term adaptation - more drastic
• What is the short term adaptation for regulating cytokines?

  SHP1 - has 2 SH2 domain and a phosphatase domain

  Recognises the phosphates on the JAK kinase - inactivates the kinase by removing the phosphate group

  Stop the JAK/STAT pathway
• What is the long term adaptation for regulating cytokines?

  SOCS protein - has an SH2 domain and a SOCS box

  SH2 domain binds to the phosphate groups on the kinase and receptor tail - blocks STAT SH2 domain from binding

  The SOCS box is linked to E3 ubiquitin ligase - JAK gets ubiquitinated and degraded

  Destroys the receptor - takes longer for JAK to be resynthesized so it is more long term
• Which STAT molecule binds to the JAK kinase when EPO binds?

  STAT-5
• How does IFN-gamma orchestrate viral defence?

  T cells and NK cells produce type II interferon IFN-gamma - IFN-gamma can kills virus infected cells or cancerous cells

  Autocrine (towards the infected cell) - inhibition of virus replication, apoptosis

  Paracrine (towards uninfected neighbours) - upregulation of MHC-I and NK cell activation, activates the immune response