L3 - Cell communication 2 - tyrosine kinase associated signalling pathway

where are transmembrane receptor tyrosine kinases and growth factor receptors found?

they are embedded in the plasma membrane

large hydrophobic molecules

they cannot cross the membrane so instead they bind to the extracellular portion of receptors

small hydrophilic molecules

they can pass through the membrane and interact with the intracellular receptors

structure of transmembrane protein

• extracellular domain

• transmembrane segment (lipid bilayer of plasma membrane)

• intracellular domain

extracellular domain of transmembrane tyrosine kinase receptor

• hydrophilic

• domain that binds the ligand

• often contains conserved elements like:

  immunoglobulin (Ig) like domain

  epidermal growth factor (EGF) like domain

  fibronectin type III repeats

  cysteine rich regions

transmembrane segment of transmembrane tyrosine kinase receptor

• region that spans the membrane

• made of alpha-helical structure made of hydrophobic amino acids

• stabilised by interactions with fatty acid chains of the lipid bilayer

intracellular domain of transmembrane tyrosine kinase receptor

• hydrophilic

• domain inside the cell that interacts with downstream signalling machinery

• in receptor tyrosine kinases (RTKs) this involves a tyrosine kinase domain - which adds phosphatase groups to tyrosine residues (tyrosine —> phosphotyrosine)

what feature in the gene sequence directs newly made transmembrane proteins to the endoplasmic reticulum, and what role does the ER play?

• the gene includes a signal peptide that directs the protein to the endoplasmic reticulum (ER)

• the ER helps insert the protein into the membrane and direct it correctly

name the two ways an extracellular ligand can trigger an intracellular response

1. conformational change in multipass transmembrane receptors

2. dimeristaion/multimerisation of single pass receptors

explain conformational change in multipass transmembrane receptors as a way of triggering an intracellular response

• these receptors pass through the membrane multiple times

• the ligand binding causes a shape change

• this conformational change is then transmitted across the membrane

explain dimerisation/multimerisation of single pass receptors as a way of triggering an intracellular response

• these receptors span the membrane once

• ligand binding causes two/more receptors to come together (dimerise)

• dimerisation activates the intracellular domains

what are single pass transmembrane proteins?

• a family of 58 single pass transmembrane proteins that span the lipid bilayer once

• they have a transmembrane domain (hydrophobic a-helix) which anchors the protein in the membrane

• they have a tyrosine kinase domain in the cytoplasmic region (inside the cell)

what is an example of a single-pass transmembrane receptor?

receptor tyrosine kinases (RTKs) are single pass transmembrane receptors that have a tyrosine kinase domain

what are types of receptor tyrosine kinases (RTKs)?

• Epidermal growth factor receptors (EGFR)

• Insulin receptor (IGF-1R)

what is the key feature of RTKs?

• the tyrosine kinase domain has enzymatic activity

• the tyrosine kinases act in trans - one receptor in the dimer phosphorylates the other = the receptors are brough together and phosphorylate/activate each other

what is the Ras/Raf (MAPK) signalling pathway?

The Ras/Raf pathway transmits signals from receptor tyrosine kinases (RTKs) to promote cell growth, division and survival

what are the steps of the Ras/Raf signalling pathway?

1. ligand binds RTKs → RTK dimerises and autophosphorylates

2. adaptor proteins (Grb2) bind phosphotyrosine

3. SOS (a GEF) activates Ras (GDP→GTP)

4. Ras-GTP activates Raf

5. Raf phosphorylates MEK

6. MEK phosphorylates ERK

7. ERK enters the nucleus → activates transcription factors → gene expression

—> as a result this regulates proliferation, differentiation, and survival

how is Raf negatively regulated before activation?

Raf is kept inactive by:

• Its own N-terminal regulatory region

• Binding of the 14-3-3 protein

• own inhibitory phosphorylation
  

  All of these must be removed or modified before Raf can be activated.

How does Erk contribute to negative regulation of the Ras pathway?

Activated Erk can negatively phosphorylate SOS, preventing SOS from binding to Grb2 (adaptor protein), which blocks further Ras activation

—> a form of feedback inhibition

What negative regulators are recruited by activated PDGFR (Platelet-derived growth factor receptor)?

Activated PDGFR recruits:

• RasGAP (which inactivates Ras by promoting GTP hydrolysis)

• Phosphatases (which remove phosphate groups from pathway proteins)

• Triggers endocytic internalisation of the receptor, reducing signalling

what are small GTPases?

• small proteins that act as molecular switches inside the cell

• they are activated when bound to GTP

• they are inactivated when bound to GDP

• part of the Rho family and Ras

what is the activity of small GTPases controlled by?

• GEFs and GAPs

• GEFs (Guanine Nucleotide Exchange Factors) help small GTPases exchange GDP for GTP - turning them ON (active)

• GAPs (GTPase Activating Proteins) speed up GTP hydrolysis (GTP → GDP) - turning them OFF (inactive)

what is the disease associated with abnormal RTK signalling (Ras/Raf/MEK/ERK)?

• cutaneous melanoma (type of skin cancer)

• often involves abnormal activation of Ras/Raf/MEK/ERK signalling pathway

• around 80% of melanomas show abnormal activation of this pathway

what is the most common cause of cutaneous melanoma?

• activating mutations in genes like Ras or BRAF

• most of the BRAF mutations occur in the kinase activation loop

• V600E mutation causes BRAF to be constantly active meaning it signals without stopping = driving uncontrolled cell growth

• this leads to increased kinase activity + contributes to melanoma development

what is the treatment for malignant melanomas?

• BRAF inhibitors - inhibitors of BRAF kinase activity

what was the issue of BRAF inhibitors?

• these drugs showed significant initial responses by shrinking the tumour however this was followed by tumours developing resistance to the drugs - resulting in disease relapse and progression

what are newer more advanced treatments for malignant melanomas?

• newer BRAF inhibitors like Vemurafenib and Dabrafenib have more selectivity for mutant BRAF and reduced off-target effect

• to improve treatment - patients are given both BRAF inhibitors and MEK inhibitor (trametinib) to reduce resistance and improve outcomes

• immunotherapies

what are examples of single-pass transmembrane receptors that are not RTKs but still signal using associates tyrosine kinases nearby?

• cytokine receptors

• T cell receptors

• Integrins

what is the JAK/STAT signalling pathway?

The JAK/STAT pathway transmits signals from cytokine receptors directly to the nucleus, leading to gene transcription

JAK/STAT signalling pathway steps

1. Cytokine binds to its receptor

2. The receptor dimerises, activating JAKs (Janus kinases) bound to the receptor

3. JAKs phosphorylate each other and the receptor’s intracellular domain

4. STAT proteins (Signal Transducers and Activators of Transcription) bind to phosphorylated tyrosines via SH2 domains

5. JAKs phosphorylate STATs, causing them to dimerise

6. STAT dimers move into the nucleus and activate target gene transcription

   —> this regulates immune responses, growth, differentiation and apoptosis

what is the JAK/STAT signalling pathway important for?

• inflammation/immunity and haematopoiesis

  → there are two types of cytokines that signal through the JAK/STAT pathway:

• anti-inflammatory cytokines - supress immune system

• pro-inflammatory cytokines - stimulate immune system

what disease does abnormal JAK/STAT pathway activation cause?

myeloproliferative neoplasms (clonally derived haematological malignancies)

what is the mutation in patients that causes myeloproliferative neoplasm?

• mutation in the JAK2 gene = JAK2 V617F mutation

• this mutation causes JAK2 to be permanently ON which results in constant activation of JAK/STAT pathway leading to uncontrolled blood cell production

what are ruxolitinib and imatinib?

• kinase inhibitors

• ruxolitinib = JAK1/2 inhibitor

• they both work by blocking different kinases and stopping abnormal signalling in cancer