Basic principles in signal transduction

• Requirement for EU existence


  • Need for IC communication
  • Decisions to migrate, proliferate, differentiate and die
  • Evolution of robust sensory and effector systems
• Distinct cell types coexist within tissues
• Plasma membrane


  An effective barrier to most molecules
• To overcome plasma membrane barrier


  Evolution of receptors (transmembrane or not) that can perceive signal as from exterior
• In vitro model of wound healing requires both the receptor and the ligand
• Features of signalling systems


  • Signal generation
  • Signal transmission
  • Signal amplification and diversification
  • Signal attenuation / termination
• Examples of major families of R relevant to cancer


  • R PK
  • Cytokine Rs
  • Frizzled / Wnt Rs
  • GPCR
  • Integrin Rs
• Src


  • Rous Sarcoma Virus causes tumours but defective strains were isolated that could replicate but not transform leading to the discovery of the v-src gene
  • Transforming capability associated with v-src
  • Cellular counterpart c-src found to be a proto-oncogene
  • V-src isolated and demonstrated to possess kinase activity
  • PK can potentially phosphroylate multiple distinct substrates. Hence, explains pleiotropic functions of src
  • Src succeeds in transforming via its PK activity
• Protein phosphorylation


  • Involves the covalent attachment of PO4 to the side chains of specific aa
  • Majority of phosphorylation occurs on hydroxyl groups of serine and threonine residues
  • Protein phosphorylation conducted by PKs
• Protein tyrosine phosphorylation


  • Very rare cellular event
  • Less than 0.1% of total cellular phosphorylation
  • PSER:pTHR:pTYR = 1800:200:1
  • Profound cellular consequences
  • Growth, metabolism, migration, differentiation, survival and death
  • V-src demonstrated to be protein tyrosine kinase
  • EGFR demonstrated to be PTK
  • By 1982, PDGF-R and insulin R had been demonstrated to be PTKs
• EGFR


  • Functions as a TK
  • Has region of Homology to Src and is the region with the kinase activity
  • Large number of RTK with differences mainly in its EC region
• An altered GFR can function as an oncoprotein. ErbB oncogene from avian erythroblastosis virus recognised to be closely related to EGFR
• TK GFR altered in human tumours


  • Deregulation of R triggering
  • Mutation allows ligand-independent activation
  • GF gene can become an oncogene if it is overexpressed
• Relaying signals by R PTKs


  • R dimerisation and transphosphroylation
  • GFs induce R dimerisation in different ways
• Role of the junta membrane domain in R PTK activation


  • Juxtamembrane region keeps the receptor inactivated
  • ATP is used to cleave the juxtamembrane domain and opens the protein to allow for further phosphorylation at the activation loops
• Structure of the cytoplasmic domain of the Kit related R, FGFR1


  • 2 lobes connected by a hinge like sequence which opens and closes like a mouth. The ATP binding site is inside the mouth and the substrate binding site is just outside the aperture
  • Activation loop turns aside following phosphroylation
• HER2 and cancer


  • HER2 complexes with EGFR to cause constant activation. Drugs have been designed to prevent dimerisation:
  • Trastuzumab blocks proteoglycans cleavage of HER2 ectodomain
  • Pertuzumab block hetero-dimerisation of HER2 domain
  • Cetuximab blocks ligand binding to EGFR ectodomain
• Reversible cycle of tyrosine phosphroylation
• ErB signalling network
• Role of tyrosine phosphroylation


  • Alters enzymatic activity
  • Creates molecular docking sites
  • Influences sub-cellular localisation
  • Induces proximity between enzymes, substrates and effectors
• Domain structure of Src protein


  • SH2 domain
  • SH3 domain
• SH2 domain


  • First identified in v-FPS, v-Src and v-Abl and subsequently in Crk, PLCy and RasGAP
  • Approx 100aa in length
  • Src binds pTYR in the context of specific C-terminal residues - pTYR-Glu-Glu-Ile
  • Identified in a number of IC signalling molecules
  • Functions as a modular plug
  • Binding site for pTYR
  • Binding site for aa side chain
  • Mediate binding to PY-containing proteins
  • Select for distinct aa motifs surrounding the PY - postal codes for selective recruitment
  • Activated GFRs are decorated with specific sets of SH2-domain combining proteins which can cause different signals to be released
• Domain structure of SH3L

  Binding to
• Mechanism of Src activation


  PY 257 is a first dephosphorylayes allwimng domain to bind PGDFR
• Individual signalling protein can contain multiple modular units where the modular domains can provide specificity to signal transduction pathways


  • Intrinsic/coupled activity - PKs'
  • IC mediators - adaptor so, GTP binding proteins
  • Effector enzymes - Protein or lipid kinases and phosphodiesterases
  • Second messengers - Ca
  • Target protein > TFs
• Structure of RAS


  Signalling cycle
• PH domain


  • Binds phospholipid belayer
  • Biochemistry of lipid balances
  • Migration of PH-containing proteins to PIP3 in plasma
  • The generation of PIP3 is reversed by the phosphatase, PTEN
• PKB controlled signalling pathway


  • 1.
• Akt/PKB controls cell size


  Akt phosphorylates and inactivates TSC2 leading to an increase in protein synthesis
• How are signalling pathways ordered?
• The power of invertebrate genetics allows for ordering of signalling pathway
• Future challenges


  • Understand roles of related signalling (RAF and B-raf)
  • Gain better appreciation of negative feedback
  • Greater quantitative understanding of signalling required
  • Fuller appreciation of all post-translational modifications required
  • Better understanding of IC localisation
  • Greater appreciation of cross-connectivity and convergence needed he
• Basic principles in signal transduction

  • Module
  • Course
  • Revised
  • Date
  • Notes
  • Completed
• Requirement for eukaryotic existence: Multicellular example: Villi of the small intestine

  
• Features of signalling systems

  • Signal generation
  • Signal transmission
  • Signal amplification and diversification
  • Signal attenuation / termination
• Receptors


  Couple detection to signals
• Major families of receptors relevant to cancer

  • Receptor tyrosine kinase
  • Cytokine receptors
  • Frizzled / Wnt receptors
  • GPCR
  • Integrin receptors

  
• Peyton Rous took chicken embryo fibroblast and grew them
• Src genes


  • Viral gene discovered from Rous Sarcoma Virus defective strains
  • Transforming capability associated with v-src
  • v-Src demonstrated to possess kinase activity
  • Explains pleiotropic functions of Src
  • Src succeeds in transforming through its protein kinase activity
  • c-Src is the cellular counterpart, a proto-oncogene

  
• EGFR family


  • Functions as a tyrosine kinase
  • Region of homology to Src with kinase activity
  • Large number of receptor tyrosine kinases with differences mainly in extracellular region
  • An altered EGFR can function as an oncoprotein