lectures 9 and 10: receptor tyrosine kinases

kinase

catalyses the transfer of a phosphate group

phosphatase

catalyses the removal of a phosphate group

what are the 2 major classes of kinases?

protein kinases and lipid kinases

why can serine, threonine and tyrosine be phosphorylated?

they all contain a -OH group in their side chains which acts as a nucleophile to form a phosphodiester bond

what percentage of mammalian proteins are phosphorylated?

30%

what percentage of the human genome is kinases?

1.7%

EGF receptor

stimulates proliferation of various cell types

insulin receptor

stimulates carbohydrate utilisation and protein synthesis

IGF receptor-1

stimulates cell growth and survival

Trk A

stimulates survival and growth of some neurones

PDGF receptor (alpha and beta)

stimulates survival, growth and proliferation

M-CSF receptor

stimulates monocytes/macrophage proliferation and differentiation

FGF receptor

stimulates proliferation of various cell types, inhibits differentiation of some precursor cells; inductive signals in development

VEGF receptor

stimulates angiogenesis

Eph receptor

stimulates angiogenesis; guide cell and axon migration

re-epithelialisation/ wound healing

during the proliferation phase, keratinocytes must rapidly migrate and multiply to cover and close the wound

what in vivo study was performed to show that FGF receptors were important in wound healing?

• mice had FGF receptors 1 and 2 knocked out

• KO mice had a significantly lower percentage of woumd close and a larger wound diameter compared to the control

what in vitro study was performed to show that FGF receptors were important in wound healing?

a scratch assay was performed on a monolayer of cultured keratinocytes, tracking how fast the cells migrated to fill the gap

extracellular domain of RTKs

contains different structural motifs (like immunoglobulin and fibronectin domains) to bind specific signalling molecules

transmembrane region of RTKs

a single membrane-spanning helix that anchors the receptor in the plasma membrane. THey also contribute to stabilising receptor dimerisation

intracellular tyrosine kinase domain of RTKs

located in the cytosol, catalyses the phosphorylation onto tyrosine residues when the receptor is activated

(this can be a single uninterrupted block of amino acids or it can be split in half)

explain how some tyrosine kinase domains are ‘split in half’

a kinase insert region is inserted right into the middle of the kinase sequence, which acts as a regulatory loop. Once phosphorylated, it creates binding sites that recruit downstream signalling proteins

what are the 3 types of ways RTKs can be activated?

1. the extracellular domains of the dimerised receptor units do not directly contact each other

2. dimerisation involves interaction from both the ligand and the receptor’s extracellular domains (direct contact)

3. ligand binding exposes a hidden dimerisation loop that the receptor binds to

why do cancer cells (like in breast cancer) grow so fast?

because receptors like HER2 and EGFR constantly send out signals

what happens when we block dimerisation?

we prevent cross-phosphorylation of the tyrosines

the N and C lobes

together, they create a pocket between them that grasps ATP and coordinate the transfer of the gamma-phosphate from ATP onto target protein substrates, allowing the kinase to do its job

the activation loop

this normally blocks the active site but when phosphorylated, the addition of the negative phosphate groups cause electrostatic repulsion forcing the loop to shift and open the binding pocket

explain the EGFR asymmetric dimer model

• when EGF binds to the extracellular domain, it forces the 2 EGFRs together into an active dimer

• inside the cell, an asymmetric dimer forms where 1 kinase domain acts as the activator which pushes against the C-lobe of the receiver kinase. The juxtamembrane segment acts as a clamp to stabilise this interaction so the receiver kinase flips into its active conformation. Once activated, it can cross-phosphorylate the C-terminal residues of its partner

• this exists in a dynamic equilibrium between inactive and active forms

SH2 domain of the Grb2

binds to the phosphorylated tyrosine residues, attaching Grb2 directly to the activated receptor

SH3 domain of the Grb2

binds to the proline-rich sequences on downstream target proteins, like Sos

activation of Sos and RAS via RTKs

1. binding of ligand causes dimerisation and phosphorylation of cytosolic receptor tyrosine residues

2. adaptor proteins like Grb2 use their SH2 domains to dock onto the phosphorylated tyrosine sites on the receptor

3. the adaptor protein brings the enchange factor Sos to the inner membrane so it can help Ras exchange GDP for GTP, turning Ras on

4. Ras activates Raf, which phosphorylates and activates Mek, which then phosphorylates MAPK

5. activated MAPK and other associated downstream kinases travels into the nucleus to phosphorylate key transcription factors

how was the drosophila eye used to determine whether Ras acts up- or downstream of the sevenless (Sev) receptor?

• when the ligand, Boss, from the R8 cell binds to sev on the precursor R7 cell, it causes an induction, developing it into an R7 neuron

• when Sev was knocked out, there was no induction

• when Sev was knocked out but Ras was activated, it still led to an induction, meaning Ras activates downstream

what are the 2 possibilities when it comes to trafficking of RTKs?

1. receptors keep their tags, which are recognised and enclosed in multivesiculat bodies and delivered to the lysosomes to be destroyed

2. deubiquitinases and phosphatases strip away the tags so they can be recycled

Cbl

an adaptor protein that binds to the phosphorylated tyrosine site, which attaches ubiquitin tags onto the receptor

how is RTK trafficking determined?

by positive or negative feedback loops