Week 4 MPW Lecture 12

Question 1

What do receptor kinases and G-protein coupled receptor have in common?

Answer 1

both use a G protein as a key part of the on/off switch

Question 2

How are G-proteins switched on and off?

Answer 2

G-proteins are switched on when bound to GTP and switched off when bound to GDP.

a) The switch is turned on by dissociation of GDP and binding of GTP.
b) It is turned off by hydrolysing GTP to GDP.
c) In the cell, the concentration of GTP is about 10x higher than that of GDP.
d) Therefore, the switch can be turned on just by persuading GDP to dissociate. Most G proteins do hydrolyse GTP to GDP spontaneously, but most also use other factors:
i) Guanine exchange factor
ii) GTPase-activating protein

Question 3

What are GEFs and GAPs?

Answer 3

Factors that are used to hydrolyse GTP to GDP or vice versa
a) GEFs are used to turn on the signal, and GAPs to turn it off again.
So these are the proteins that actually turn the switch on and off.

Question 4

What method do receptor-linked kinases use?

Answer 4

Use dimerization, the kinase domains need to be dimerise to work

Question 5

What is dimerization?

Answer 5

A dimerization is an addition reaction in which two molecules of the same compound react with each other to give the adduct.

Question 6

What is phosphorylation?

Answer 6

The addition of a phosphate group

Question 7

What effect does phosphorylation have on the kinase domains?

Answer 7

Dimerization then autophosphorylation

Question 8

General steps of a receptor–linked kinase?

Answer 8

Phosphorylation of one kinase domain by the other fixes the position of the activation loop, which allows it to bind substrate correctly.

a) After phosphorylation of the receptor, the intracellular signal is considered on and acts as a binding site for modular adapter proteins (Grb2 and Sos)
i) Grb2 contains 2 types of domains:
(1) The SH3 domain recognises polyproline helices
(2) The SH2 domain recognises phosphotyrosines. There is a specific SH2 for each receptor.
ii) Sos
(1) Contains a GEF domain and a proline-rich arm (binds to SH3)
b) This activates the G protein ras
c) The Sos activates Ras and then activate RAF
d) ERK then moves into the nucleus and phosphorylates sever transcrtion factors.

Question 9

What is a kinase cascade?

Answer 9

A sequence of kinases which phosphorylate each other and amplify the sifnal and each step

Question 10

Why is it an advantage to have many sequences in a reaction chain?

Answer 10

The signal can be modified and controlled

Question 11

Why are the Switch I and Switch II regions of G-proteins called “switches”?

Answer 11

Because when they bind to GTP they act to turn on the G-protein switch

Question 12

Explain (in writing) the functions of GEFs and GAPs

Answer 12

GEFs (guanine exchange factors) catalyse the dissociation of GDP from G proteins and therefore allow GTP to bind. They thus activate G proteins. GAPs (GTPase activating proteins) catalyse the hydrolysis of GTP to GDP, and therefore turn G proteins off.

Question 13

G proteins are found in a range of functions as well as signalling. Find out two quite different such functions.

Answer 13

There are many! Some interesting ones: (a) The ribosomal elongation factor EF-Tu is a carrier for aminacyl-tRNA (ie the tRNA covalently attached to its cognate amino acid). A complex of EF-Tu.GTP and aminacyl-tRNA binds to the ribosome. If the tRNA anticodon matches the mRNA, then GTP is hydrolysed, EF-Tu is “switched off” and it dissociates, leaving the correct amino acid to be added to the growing peptide chain. (b) Myosin is the protein that binds and detaches from actin to cause muscle contraction. It does this in response to ATP binding and hydrolysis (rather than GTP) but the structure of the key ATP-recognition part is basically a G-protein. Kinesin moves along microtubules to carry vesicles and has a very similar ATP-recognition domain. (c) In lecture 5 (particularly q2) I introduced Rab proteins, which are involved in binding and detaching vesicles at the start and end of transport. These are G proteins with very similar structure to Ras.

Question 14

What is the biological function of VEGF?

Answer 14

it stimulates the growth of new blood vessels. All tissues need oxygen, which is carried by blood vessels. Newly growing tissue needs a blood supply, and the blood vessels are produced in response to VEGF. Cancer tumours also need a blood supply, stimulated by VEGF, so VEGF inhibitors are in use as anticancer drugs.

Question 15

If the kinase domain in an unactivated receptor is inactive, how does it manage to autophosphorylate?

Answer 15

it is not completely inactive, it is just not a very good enzyme, and will only act as a kinase if the substrate is forced into the active site, eg on receptor dimerisation.

Question 16

List the modular signalling domains found in the VEGF pathway, an describe their functions. In what way are they “modular”? (You will need to do some background reading on this)

Answer 16

The signalling domains I described are:
SH3. This domain recognises specific polyproline sequences
SH2. This domain recognises phosphotyrosine.
If you wanted to you could call the kinase domain another modular signalling domain.
They are modular because they can be genetically transplanted into other signalling pathways and still have very similar functions – so for example if you put an SH3 domain into an existing signalling molecule, then it will be able to recognise and bind to polyproline sequences. This is what I was referring to in lecture 1 – a modular system (like a modular kitchen!) is the easiest way to create a new function without having to start from scratch every time.

Question 17

The generic names for the kinase cascade enzymes is MAPK/MAPKK/ MAPKKK, where MAP stands for Mitogen Activated protein Kinase. What is a mitogen, and what is the link between mitogens and signalling?

Answer 17

A mitogen is a chemical that causes cancer. It was observed that when you treat cells with mitogens, this turned on a range of kinases. It was eventually realised that what the mitogens were doing was turning on signalling pathways and that the kinases were part of the pathways. A large number of cancers involve over-activity of signalling pathways (often the failure of a signalling pathway to be turned off).