Week 6/7: Receptor Tyrosine Kinases (RTK)

• receptor tyrosine kinases (RTK) are eukaryotic membrane bound receptors that has

  • N terminal extracellular domain -> binds specific substrates and responds to extracellular protein/peptide hormones
  • transmembrane helix
  • C terminal cytoplasmic domain -> tyrosine rich kinase tail that auto phosphorylate and phosphorelay that to other targets

  there are 58 different genes and thus RTK in eukaryotes

  
• the different molecules that RTK respond to are

  • mitogen -> small peptide/protein that induces a cell to begin cell division
  • mitogenesis -> triggering of mitosis, typically through activation by a mitogen
  • growth factors (GF) -> mitogen/mitogen like molecules that can induce varying effects on the cell
  • growth, proliferation, motility, survival, protein synthesis, autophagy, transcription
  • *GF can act as mitogens but mitogens can't be GF
• how ligand binding to RTK cause signalling, level 1 change

  1. ligand (GF) binding to receptor -> kinase activation and auto phosphorylation
  2. phosphorylated RTK recruit specific cytosolic proteins
  3. recruited proteins get phosphorylated -> cause activation

  
• step 1: ligand binding causes activation of RTK

  • RTK are monomeric while inactive, ligand binding induces
  • conformation change
  • dimerization of receptors -> activates kinase domain
  • 2 ways to phosphorylate, kinase domain:
  • auto phosphorylate
  • trans phosphorylate
  • **can be done on multiple residues, each residue site recruits a specific adaptor protein

  
• ligands involve mitogens, growth factor, hormone and are interchangeable for RTK
• the 2 ways that ligand binding causes receptor dimerization is:

  • ligand binding induces conformational change (disrupt noncovalent interactions) that induce dimerization
  • for ex. a new hydrophobic interface is exposed that needs to bind to another hydrophobic interface to protect it
  • divalent ligands -> cause receptors to dimerize upon binding
  • ligands are divalent or form dimers at specific concentrations
  • are symmetrical about its interface

  
• step 2: recruitment of signalling proteins to phosphorylated receptors

  • after ligand binding event -> RTK are phosphorylated on multiple TYROSINE residues -> each act as docking site for another protein in cytosol which usually have an SH2 or PTB domains that facilitate their interaction
  • first binding site -> specific for phosphotyrosine
  • second binding site -> specific for another amino acid (specificity marker)

  
• important domains for RTK signalling

  • Src Homology 2 (SH2) and PhosphoTyrosine Binding (PTB) domain binds to unique motifs in proteins ONLY when a key tyrosine residue is phosphorylated (pTyr) and a residue in close proximity (for specificity)
  • both play large role in modular nature of signals and most proteins contain these domains
  • SH2 is distinct because these domains have a conserved sequence and structure
  • these domains are usually part of a larger domain containing auxiliary domains serving another function
• the epidermal growth factor receptor (EGFR) controls various aspects of cell physiology like proliferation, survival, migration, and metabolism (autophagy)
• EGFR is key for development and homeostasis of adult tissues

  
• key players in the MAPK (Ras/Raf/Erk) pathway

  • EGF -> monomeric ligand that stimulates cell growth and proliferation
  • EGFR -> RTK activated by EGF binding, induce conformation change -> dimerization
  • Grb2 -> adaptor protein containing SH2 domain
  • Sos -> GEF for Ras GTPase
  • Ras -> GTPase
  • Raf -> Ser/Thr protein kinase, MAP3K
  • Mek -> Tyr/Thr protein kinase, MAP2K
  • Erk -> Ser/Thr protein kinase, MAPK
• monomeric EGF ligand binds EGFR which changes conformation and causes dimerization of receptor which auto/trans phosphorylate on tyrosine rich C terminal tail in ATP dependent manner

  
• MAPK pathway

  • EGF binds EGFR -> conformation change -> dimerization of EGFR, kinase domain activates -> auto/trans phosphorylation of C terminal tail -> phosphotyrosine site binds Grb2 at SH2 domain -> SH2 binds Sos as SH3 domain -> Sos does GEF function on Ras GTPase -> Ras activates Raf MAP3K -> Raf allosterically phosphorylates Mek MAP2K -> Mek allosterically phosphorylates Erk MAPK -> Erk phosphorylates other targets in cell
  • need at least 4 ATP to turn on 1 protein target

  
• Mek can be allosterically phosphorylated at 2 sites which makes it more active, Mek can allosterically phosphorylate Erk at 2 sites which also increases activation

  
• Ras is one of the most frequently mutated gene in human cancers -> Ras on = cell proliferation, migration, etc. -> no negative feedback -> cancer

  
• phosphatidylinositol (PI) is a glycerophospholipid with an inositol sugar headgroup which can be phosphorylated on 3 positions at their hydroxyl groups by a lipid kinase in response to specific cues to become PIP (s)
• key players in the PI3K/Akt/mTOR pathway

  • GAB -> adaptor protein
  • PI3K -> Ser/Thr kinase for PIP2
  • PIP2 (PI(4,5)P2) -> minor phospholipid in membrane
  • PIP3 -> PI(3,4,5)P3, second messenger localized at membrane
  • PDK1 -> Ser/Thr kinase, phosphorylates Akt
  • mTOR -> Ser/Thr kinase
  • PTEN -> protein tyrosine phosphatase (PTP), acts on PIP3
• The PI3K/Akt/mTOR pathway

  monomeric EGF binds EGFR -> dimerization of EGFR, kinase domain activated -> auto/trans phos at C terminal tail on tyrosine residues -> GAB binds to pTyr residue on EGFR by its SH2 domain -> GAB recruits PI3K -> PI3K phosphorylates PIP2 to make PIP3 -> PIP3 recruits PDK1 and Akt -> Akt phosphorylated by 2 proteins => PDK1 and mTOR -> once Akt is di phosphorylated it is released by PIP3 and active as terminal kinase to activate other targets

  • PTEN is a PIP3 phosphatase (PTP) which removes terminal phosphate to return to PIP2

  
• activated Akt is released from PIP3 once phosphorylated by PDK1 and mTOR where it changes cellular processes leading to survival and growth
• epithelial cells are the surface layer of your body and organs, serves as barrier between outside and inside of your body as well as protect from harmful agent

  
• carcinomas are cancers specific to epithelial cells and disrupt intercellular tight junctions and cause alterations in cell polarity, they grow abnormally (not in normal monolayer)

  
• invasive cancer occurs when epithelial cells depart from local tissue to invade and occupy other parts of local tissue/organ

  • mutated cell -> hyperplasia -> dysplasia -> in situ cancer -> invasive cancer (enters blood vessels)

  
• epithelial mesenchymal transition (EMT) refers to a regulated change of state of an epithelial cell to become a mesenchymal cell (migratory, develop invasive properties, and can remodel the extracellular matrix)

  • EMT is detected by gain/loss of certain proteins (ex. LOSS of E-cadherin)
  • epithelial cell loses normal cell polarity (apical/basal) due to loss of tight junctions -> changes cell morphology, ECM, increases cell motility

  
• mesenchymal cells are migratory, develop invasive properties, and can remodel the extracellular matrix, EMT is the transition from epithelial to mesenchymal cell
• EMT occurs are specific key times during normal development like embryogenesis but in cancer, EMT occurs when it's not supposed to -> cells become migratory and invasive (EMT is not a new function by cancer!)

  
• EGFR activates many signals/switches (can be sequential/parallel) and controls many cell outcomes, these signals don't always stay on/activated so there are negative feedback pathways put in place which is a target for cancer cells to shut off
• 3 negative feedback mechanisms for MAPK pathway

  • GTP to GDP -> Ras GTPase hydrolyzes from GTP to GDP -> inactivates pathway
  • mutation in Ras that inhibits hydrolysis ability promote cancer
  • phosphorylating upstream -> Erk can phosphorylate proteins upstream to inactivate such as Mek or Raf at an allosteric site (inhibits kinase activity) or EGFR
  • phosphorylated EGFR -> EGFR is phosphorylated at its Tyr residue which becomes targeted for ubiquitination to get degraded in proteosome, pErk can phosphorylate EGFR at a Ser/Thr residue to help ubiquitination
• once EGF binds to EGFR -> Erk will phosphorylate EGFR which causes ubiquitination by ubiquitin ligases (removed by DUB) -> endocytosed into cell

  
• RTK have a C terminal Tyr rich domain that is phosphorylated on many sites which have surrounding residues of a defined chemical charge/structure that are specific for certain adaptor proteins containing domains like SH2 or PTB