Microbiology 18: (Tanaka) Regulatory mechanisms to define signalling output

Question 1

Mechanism of RTK signalling in Ras and MAPK cascade? Proteins involved?

Answer 1

Activated RTK has high affinity phosphotyrosine docking sites revealed

• > recognised by SH2 domain of adaptor protein Grb2
• > Ras-GEF protein Sos is bound to Grb2 via SH3 domains

-> Sos acts as a GEF catalysing exchange of GDP in inactivated Ras proteins for GTP

GTP exchange activates Ras protein

Ras further signals downstream

Question 2

What is Ras? What switches on and off these types of molecules?

Answer 2

Monomeric GTPase (small G-protein)

• > switched on via GEFs (Guanine nucleotide exchange factor)
• > switched off via GAPs (GTPase activating protein)

Question 3

Downstream ras signalling? Molecules involved?

Answer 3

Serine-Threonine kinases

MAP kinase kinase kinase (Raf)

MAP kinase kinase (Mek)

MAP kinase (Erk)
-> transcription factors / changes in protein activity

Question 4

How do differences in ERK activation affect cellular responses? WHy?

Answer 4

Sustained ERK activation
and
Transient ERK activation

have different effects in the same cells

• Regulatory mechanisms are important to shape signalling activation profiles and localisation

Question 5

Levels of regulation in these protein mechanisms?

Answer 5

Protein degradation
Protein translocation
Protein de-phosphorylation by phosphatases
Protein phosphorylation by kinases

Question 6

TNF-R / NF-KappaB pathway? How does it affect gene expression?

Answer 6

1) Ligand - TNF-alpha binds to TNF receptor

2) Initially activates molecule IKKK -> becomes IKKKa
(kinase)

3) further activates IKK -> becomes IKKa
(kinase)

4) finally activates transcription factor NF-KappaB, enters nucleus via NLS
(TF) -> regulates many gene expressions

5) Gene expressions include (negative feedback)
- IkappaBalpha -> inhibits NF-KappaB
- A20 -> inhibits IKKK, IKK
- Response proteins

Question 7

Differences in signal between kinases within:

No feedback
Positive
Negative short delay
Negative long delay

Answer 7

Neutral -> signal rises and remains constant at peak, ends when signal kinase is no longer expressed

Positive -> Signal rises and overall amplitude increases as activated kinase works to further activate itself -> signal sustained but at lower amplitude after signal kinase expression ends

Negative short delay -> Signal rises but is counteracted shortly after by activated kinase inhibiting further activation of itself -> lower subsequent amplitude sustained once cell establishes equilibrium

Negative long delay -> amplitude fluctuates up, and down after delayed negative feedback from activated kinase (sustained oscillations as long as stimulus present)