JAK/STAT Signalling Flashcards
Explain growth hormone synthesis
- Mature peptide is 191 amino acids long with molecular weight of 20kDa.
- Synthesised as a 217 amino acid pre-protein (191 amino acids + 27 amino acid signal peptide).
- Signal peptide - anchors pre-protein to ER membrane.
- ER membrane-bound signal I peptidase cleaves pre-protein between residues 26 and 27 resulting in the 191 residue-long mature secretory GH.
Describe the structure of Growth Hormone
Mature secretory hormone consists of single polypeptide chain that folds into a four α-helix bundle.
Glycine at postion 120 is essential for growth hormone function
Describe Growth Hormone Receptor
Belongs to the Cytokine Class 1 Receptor family that shares common features:
Extracellular Domain:
- 4 conserved cysteine residues
- WSXWS motif
Spans the phospholipid bilayer
Intracellular Domain:
- Region that binds JAK proteins
- NO INTRINSIC KINASE ACTIVITY
Describe other class I Cytokine Receptors
•Homomeric receptors (subunits are identical):
- growth hormone
- Leptin
- erythropoietin
- thrombopoietin
Describe multimeric receptors (subunits are different)
Shared b: IL-3, IL-5, GMCSF
Shared gp130: IL-6, IL-11, IL-27, IL-31, CNTF, CT-1, LIF
Shared g: IL-2, IL-4, IL-7, IL-9, IL-15, IL-21
Describe Growth Hormone Receptor Activation
Binding of Growth Hormone to its receptor induces receptor activation via ligand-mediated oligomerisation
Describe JAKs (Janus Activated Kinases)
- 120-140 kDa in size
- seven Janus homology domains (within the N-terminal region of the protein and allow the JAK proteins to join to the receptor
- JH1 domain at the C-terminal region and contains the enzymatic kinase activity of the protein
- JH2 pseudokinase domain that regultes kinase activity
Explain how the activation of the growth hormone receptor induces a series of phosphoylation events inside the cell
- Protein Phosphorylation in reversible Post-Translational Modification (PTM) of proteins
- Protein kinases mediate addition of phosphate group at serine, threonine and tyrosine side chains (ATP dependent).
- Protein phosphatases reverse protein phosphorylation by hydrolyzing the phosphate group.
- Phosphorylation changes activity of target protein (both negatively and positively).
How can we monitor the phosphorylation of JAX in living cells?
- 3T3 F442A (pre-adipose) cells.
- Exposed to hGH.
- Western blot of protein extracts.
- Antibody only recognises phosphorylated tyrosine residues
From the image:
When probing JAK proteins for phosphoylation, when the proteins are treated with human growth hormones, you can detect the species of JAK proteins in the western blot
Describe Signal Transducer and Activator of Transcription (STAT)
- Coiled-coil domain at the N-terminal domain (important for transcriptional activation)
- C-terminal tyrosine (important for phosphorylation)
- SH2 domain mediates interactinos between different proteins
Explain SH2 Domains
- Found in wide range of signalling molecules.
- SH2 domains allow proteins to bind to phosphorylated tyrosine residues on other proteins.
- Commonly found in adaptor proteins that aid in the signal transduction of receptor tyrosine kinases.
- Localise signalling proteins together to generate signalling hubs.
Describe Signal Transduction: Decsribe the generic JAK/STAT signalling pathway
- Binding of the ligand to the receptor induces receptor dimerisation
- This brings their associated JAK proteins in close proximity and they can transphosphorylate
- This increases the kinase activity of the JAK proteins so they phosphorylate tyrosine residues within the receptor tail itself
- The phosporylated tails act as binding sites for the STAT proteins through their SH2 domains
- JAK proteins phosphorylate tyrosine residues within the STAT proteins
- Once the STAT proteins are phosphorylated they dissociate form the membrane and they dimerise
- This dimerisation event exposes a nuclear localisation signal
- The STAT dimers can now enter the nucleus where they bind to specific DNA sequence to activate gene expression
Summary: generic JAK/STAT signalling
- Ligand (growth hormone) binds to receptor and induces receptor dimerization.
- Growth hormone receptor has no intrinsic kinase activity.
- Receptor dimerization brings JAKs in close proximity – trans-activate.
- JAKs phosphorylate specific tyrosine residues in cytoplasmic tail of receptor.
- Phosphorylated tyrosines act as binding sites for SH2 domains of STAT proteins.
- •JAKS phosphorylate bound STATS.
- •Released from receptor and dimerise – exposes nuclear localisation signal.
- STAT dimers enter nucleus, bind to enhancers and activate gene transcription.
STAT Nuclear Localisation in response to growth hormone stimulation
GFP-labelled (flourescent tag) STAT5 translocates to the nucleus after GH stimulation
In control cells the flourescent protein is localised throughout the cell
Upon stimulation with a with a growth hormone the flourescent STAT protein becomes localised within the nucleus
Describe Transcriptional Regulation by STATs
- Nuclear STAT binds to a consensus DNA-recognition motif.
- gamma-activated sites (GAS).
- promoter region.
- activates transcription.
Example: transcription of IGF-I
IGF-I mRNA levels increase in response to GH stimulation
Timed intervals after growth hormone stimulation shows IGF-1 mRNA levels increase
Switching Off Signalling-Short term regulation:
JAK deactivation by SHP1 phosphatase (SH2 and phosphatase domain) that allows it to bind the phosphorylated tyrosine residues in the activated tail. The phosphatase domain hydrolyses the phosphate to inactivate the JAK protein
Switching Off Signalling-Long term regulation:
Protein degradation by SOCS proteins (negative feedback)
- SOCS proteins have an SH2 domain so they can bind to the phosphorylated tyrosine residues in the recepor tail
- They also contain a domain that can recruit E3 ubiquitin ligase to target JAK proteins for degradation via the proteosome
Describe the JAK/STAT signalling pathway in relation to IGF-1 expression
In response to growth hormone stimulation IGF-1 is produced
IGF-1 negatively regulates the production and release of IGF-1 from the anterior pituitary
Describe Growth Hormone Deficiency
- There is less growth hormone available to bind to the receptor therefore it doesnt dimerize and become activate.
- There is an absence of JAK kinase activity, no binding sites availble for SH2 domains of STAT proteins.
- No STAT protein entry into the nucleus therefore no induction of target gene expression
What happens to the signalling cascade when growth hormone levels are high- Acromegaly?
- Abundance of growth hormone
- The pathway is constitutively
- High levels of expression of target genes, including IGF’s
- Normal GH regulatory mechanisms are absence
