W17 Flashcards
Describe the catalytic domain of kinases.
N-lobe and C-lobe with an ATP and substrate binding pocket between the two lobes, within the binding pocket is an activation loop that activates the kinase upon phosphorylation
Describe SH2 domains of tyrosine kinases.
Bind to phosphorylated tyrosine residues, third residue downstream of Tyr determines the specificity of a particular SH2 domain
Describe SH3 domains of tyrosine kinases.
Bind to proline residues found in a type II poly-proline helix, specificity of an SH3 domain is determined by residues between the Pro residues
What is the difference between c-Src and v-Src?
c-Src has two tyrosine phosphorylation sites: Y419 activation loop and C-terminal Y530, v-Src only has Y419 phosphorylation site
What is the significance of the Y530 phosphorylation site in c-Src?
Phosphorylated Y530 forms a binding site for c-Src’s own SH2 domain, creating an intramolecular interaction which creates a ‘hairpin’ structure that sequesters the catalytic domain, preventing it from accessing its substrate
Why is v-Src lacking Y530 significant?
Unable to form the inhibitory hairpin so is constitutively active and promotes cell proliferation seen by cancers
Describe the effect of epidermal growth factor (EGF).
EGF binds to EGF receptor and triggers phosphorylation of multiple cellular proteins, causing cell proliferation
Describe the structure of the EGF receptor (Her/ErbB) family.
Intracellular tyrosine kinase domain and two extracellular cysteine-rich domains. Four different Her/ErbB family members in humans, ErbB2 cannot bind EGF so its dimerisation arm is always exposed, ErbB3 has no kinase activity, v-Erb has no extracellular domain so cannot be inhibited and remains constitutively active
Describe the effect of EGF binding to EGFR.
EGF binds, causing a conformational change that exposes the dimerisation arm, two EGFR dimerisation arms associate with eachother, causing in trans autophosphorylation to activate the kinases to autophosphorylate other Tyr residues on the cytoplasmic region
Describe the effect of Herceptin on ErbB2 EGF receptors.
Monoclonal antibody binds to the extracellular portion of ErbB2, inhibiting dimerisation and internalising the receptor from the plasma membrane
Describe how EGF receptors activate Ras.
EGF binds to EGFR, causing dimerisation and in trans autophosphorylation, activating kinase phosphorylation of the cytoplasmic region, SH2 domain of Grb2 anneals to pTyr and SH3 domains of Grb2 bind to poly-proline sequences on the GEF Son of sevenless (Sos) which exchanges Ras GDP for GTP, activating Ras conformational change to expose Switch I and Switch II to interact with effector molecules
Describe two ways Ras is regulated.
GTPase activator protein (GAP) such as NF1 inserts an arginine finger into the Ras GTPase region to increase catalysis of GTP. Ras GDIs sequester Ras away from the plasma membrane by binding to the Ras lipid tail that would otherwise be integrated into the plasma membrane
Describe the effector molecule cascade caused by Ras.
Ras Switch I and Switch II interact with the Ser/Thr kinase Raf. Raf is a MAP kinase kinase kinase which phosphorylates the Thr/Tyr kinase MEK. MEK is a MAP kinase kinase which phosphorylates the Ser/Thr kinase ERK. ERK is a MAP kinase which phosphorylates TCFs, which adopt an active conformation that binds to promoters for genes required for cell proliferation. ERK also causes negative feedback by phosphorylating the tyrosine kinase domain of the EGFR and phosphorylating Sos to prevent interaction with Grb2
