Block A Lecture 4: Tyrosine Kinases

Question 1

What kind of polypeptide is the insulin receptor?

Answer 1

An α2ß2 polypeptide
(Slide 4)

Question 2

What kind of bonds is the insulin receptor polypeptide held together by?

Answer 2

Disulphide bonds
(Slide 4)

Question 3

Outline where the alpha and beta subunits of the insulin receptor residue in relation to the cell membrane and the phospholipid bilayer.

Answer 3

The alpha subunits are located outside of the cell and connect to a small portion of the beta subunit which also residues outside the membrane.
The beta subunit is trans-membrane, with a majority of it being located within the cytosol
(Slide 4)

Question 4

How is a signal transported across a membrane by the insulin receptor?

Answer 4

1. Insulin binds to the α2 subunits.
2. These then transmit the signal to the ß2 subunits
3. This activates an intrinsic tyrosine kinase within the cytosolic domain of the ß-subunit

This shows the signal has crossed the membrane
(Slide 4)

Question 5

How does the insulin receptor form a signalling complex?

Answer 5

After it becomes activated, the intrinsic tyrosine kinase of the receptor phosphorylates specific Tyr residues within the receptor. (This is known as auto-phosphorylation)
These p-Tyr residues then recruit signalling molecules to the receptors which are the themselves phosphorylated.
This forms a signalling complex
(Slide 5)

Question 6

How can a ligand binding activate a receptor via trans autophosphorylation?

Answer 6

1. A ligand (such as EGF or insulin) binds to the extracellular domain of the receptor.
2. The ligand binding induces a conformational change in the receptor which promotes dimerization
3. Dimerization brings the intracellular kinase domains of the two receptors closer together which allow them to act on each other
4. The kinase domains on the cytoplasmic sides of the receptors phosphorylate specific tyrosine residues on the other receptor in the dimer (this is called trans-autophosphorylation)
5. This induces conformational changes which fully activate the kinase activity of the receptor

(Slides 7, 8 and 9)

Question 7

What is unusual about the trans autophosphorylation of insulin?

Answer 7

Insulin already exists as a pre-formed dimer, even without any ligands binding to it, so when insulin binds it doesn’t promote dimerization, it causes a conformational change instead, which activates the tyrosine kinase activity of the ß subunits and brings them close enough for them to undergo trans-auto-phosphorylation
(Slide 7)

Question 8

How can we analyse how the insulin receptor changes structure in response to insulin bonding?

Answer 8

By incorporating the receptor into an artificial system and look at how its structure changes upon insulin addition using high resolution cyro-electron microscopy
(Slide 11)

Question 9

How does the conformational change resulting from a ligand binding to a receptor and promoting dimerization lead to the activation of the intrinsic tyrosine kinase?

Answer 9

As the conformational change results in an activation loop near the active site of the tyrosine kinase moving, enabling substrates to bind to the active site
(Slide 16)

Question 10

How does phosphorylation occur in trans auto-phosphorylation?

Answer 10

Chain A phosphorylates a tyrosine in chain B and then that phosphorylates a tyrosine in chain A… etc

(Slide 20)

Question 11

What are phospho-tyrosines?

Answer 11

Tyrosine residues which have been phosphorylated
(Slide 22)

Question 12

What do phospho-tyrosine residues act as?

Answer 12

A magnet for key signalling molecules
(Slide 22)

Question 13

What are SH2 domains?

Answer 13

A protein domain found in many signalling proteins
(Slide 22)

Question 14

What do SH2 domains bind?

Answer 14

Phospho-tyrosine residues
(Slide 22)

Question 15

What does a SH2 domain binding to a phospho-tyrosine do?

Answer 15

It recruits the SH2 domain containing protein (a signalling molecule) to the receptor.
(Slide 23)

Question 16

What occurs after the SH2 domain-containing protein is recruited to the vicinity of the activated receptor?

Answer 16

The SH2 domain-containing protein is then activated, enabling it to propagate the signal
(Slide 23)

Question 17

What do all SH2 domains have?

Answer 17

A “pocket” which the phospho-tyrosine fits into
(Slide 25)

Question 18

Not all SH2 domains bind all P-tyrosine residues. How is the specificity for this dictated?

Answer 18

By the surrounding sequences in the target polypeptide (the sequences surrounding the tyrosine)
(Slide 25)

Question 19

Do SH2 Domain containing proteins contain other domains?

Answer 19

Yes, often
(Slide 29)

Question 20

How can a single effector have 2 distinct functions in 2 different cell types?

Answer 20

Different receptors assemble different protein complexes depending on which effectors are recruited. This in turn dictates which proteins are expressed in a given cell type. Therefore a single receptor can have 2 distinct functions in 2 cell types as the downstream effectors are different
(Slide 30)

Question 21

How is phospholipase-gamma activated by receptor tyrosine kinases?

Answer 21

Phospholipase-gamma is recruited to the receptor via the specific SH2 domain.
This brings Phospholipase-gamma into close proximity to the tyrosine kinase domain of the receptor.
Phospholipase-gamma is then phosphorylated and activated
(Slide 37)

Question 22

How is the MAPK protein kinase activated?

Answer 22

Ras-GTP activates the signalling pathway.
It’s activation is controlled by the balance of Ras-GTP and Ras-GDP controlled by the relative activity of Ras-GAP and SOS proteins

Note: Recruiting and activating these to receptors via SH2 domains is a key control step of this cascade
(Slide 38)