What acts on segment boundaries established by bicoid and its inhibitor hunchback?
Segment polarity genes Hh and Wnt
Describe what happens in the JAK/STAT pathway when GH/cytokine stimulates a RTK
JAKs bind to a RTK and phosphorylate each other.
STAT binds pY via its SH2 domain.
This acts as a transcription factor
How is a STAT signal sequestered?
Phosphatase or SOCS block the SH2 domain.
2 phosphorylated STATs can bind to each other and they are ubiquitinated and degraded.
Describe the Wnt signalling pathway
- Wnt activates the GPCR frizzled.
- This activates the GPCR dishevelled
- Dishevelled inhibits GSK3/APC/Axin
- β-catenin cannot be phosphorylated, ubiquitinated and degraded so it acts as a transcription factor.
How many Frizzled receptors have to be stimulate to induce a Wnt response?
2
What is Wnt signalling involved in?
Osteoblast formation
Stem cells
Overexpressed in mammy cancers
What type of signalling is Notch/Delta?
Contact dependent that works by lateral inhibition
Describe how Notch activates its receptor Delta
- Notch is synthesised in the ER
- Proteolytically cleaved in golgi but subunits remain non-covalently joined
- Notch moves to nucleus and binds Delta
- ADAM10 cleaves the notch extracellular domain
- Y-secretase cleaves the notch cytosolic segment which moves to the nucleus and acts as a transcription factor
Describe NF-kB signalling?
- Toll-like receptors or cytokine receptors are activated
- I-kB kinase phosphorylates 2 Ser residues on I-kB, so that E3 ubiquitin ligase ubiquitinates and degrades it.
- The NF-kB NLS is exposed so it shuttles to the nucleus and upregulates inflammatory genes
What does Hhg signalling regulate?
Adult stem cell growth
Hair follicle growth
Salamander limb regeneration
How is Hh signalling repressed?
Without Hh, Ci clusters onto microtubules, are phosphorylated, ubiquitinated and proteolytically cleaved.
The N-terminal product repressed Hh target genes
How is Hh signalling involved in moving cilia?
- When Smo is inhibited by BARK and β-arrestin, motor protein Kif7 joins it to the microtubule and zips it to the cilary membrane
- Smo activates the Gli transcription factor.
What is the Gli transcription factor involved in?
Cell fate
Proliferation
Patterning
Outline the Hh signalling cascade?
- Hh binds to Patched GPCR which is then endocytoses and degraded
- Smoothened is no longer inhibited by Ptc, so moves from internal vesicles to the plasma membrane
- Smo is phophorylated and assumes an open conformation which increases phosphorylation of the Fu and Cos2 complex
- Fu, Cos2 and Ci complex dissociates from microtubules
- Cos2 binds phosphorylated Smo, disrupting the complex
- Ci moves ti the nucleus and activates CREB binding protein, upregulating Hh genes
What properties should you look out for drugs
ADME
Lipinski’s rule of 5
Selective (few off target effects)
Ideally hydrophilic exterior to be soluble in bloodstream
Non-toxic
What are lipinski’s rule of 5
10< H-bond acceptors
5< H-bond donors
MW < 500kDa
logP < 5
Describe two Ras signalling cascades
Ras -> MAPKKK -> MAPKK -> MAPK
Ras -> Raf -> MEk-> ERK -> ERK-p -> fos phosphorylation -> gene activation
How does ERK move to the nucleus?
MEK5 phosphorylates it, disrupting its NES. It shuttles into the nuclear pore
Describe how Raf acts as a kinase
- S259 is phosphorylated and 14-3-3 clamps Raf shut by binding to the Ras Binding Domian (RBD)
- 14-3-3 detaches from RBD so it can bind to Ras
- Y341 is uncovered and phosphorylated, activating the PKC activation loop. Raf is activated as a kinase
14-3-3 is phosphorylated at other points
How does the Raf system act?
14-3-3 inhibits Ras
14-3-3 activates PKC
Raf system is held in a scaffold for co-localisation so functional groups interact
Linkers can be modified to alter frequency of collisions
