Lots of kinases (L12)

Question 1

What is the function of Ras?

Answer 1

A small membrane protein who key function is to relay signals for cell differentiation and proliferation, it is a key ‘switch’ component of a large variety of signalling pathways

Question 2

What does the Ras-MAP kinase pathway do?

Answer 2

Activates genes involved in cell proliferation

Question 3

What is Ras regulated by?

Answer 3

GEF (guanine nucleotide exchange factor) and GAP (GTPase activating protein)

Question 4

What is the Src family?

Answer 4

Receptor-associated tyrosine kinases involved in diverse functions including B and T cells and are anchored to the inner membrane surface

Question 5

What is the structure of inactive Src tyrosine kinase?

Answer 5

Helix C
Gly flap
Activation loop
SH2- phosphorylated tyrosine binding
SH3-protein binding which is rich in proline

Question 6

How may a tyrosine kinase be activated?

Answer 6

pTry activates via activation loop
Tyr527 is phosphorylated in the kinase domain which inactivates the SH2 pTyr recognition site keeping it in an inactive conformation
Linker occupies SH3 protein binding site. Binding of target molecule by SH2 and/or SH3 may free kinase domains to adopt active conformation

Question 7

What may be required to keep tyrosine kinase in its inactive form?

Answer 7

N-terminus myristate bound to the C-terminal kinase domain
src N-terminus cleaved before crystallisation
Catalytic residues, the helix C Glu310, the gly flap are in inactive conformation

Question 8

How is Ras activated via the Src family in TCR-mediated signalling?

Answer 8

1. Src-type receptor associated tyrosine kinase Lck creates pTyr sites on ITAMs of co-receptors
2. Lck phosphorylated ITAM pTyr sites act as SH2 docking sites for the key protein kinase ZAP-70. ZAP-70 then phosphorylates and activates other signalling components leading to activation of signalling pathways including that mediated by Ras.

Question 9

How is Ras activated via the Src family in BCR-mediated signalling?

Answer 9

1. Src-type kinases activated by interaction with the ITAMs, create pTry docking sites on ITAMs for SH2 domains of the key protein kinase Syk
2. Syk is then activated by phosphorylation and goes on to phosphorylate and activate other signalling molecules including the adapter protein BLNK
3. Activated adapter protein BLNK displays SH2 docking sites for Btk and PLCgamma2
4. Syk then phosphorylates and actiavtes the complexed Btk
5. Btk then phosphorylates and activates PLCgamma2, leading to B-cell activation via signalling pathways including that mediated by Ras

Question 10

How is Ras activated via receptor tyrosine kinases?

Answer 10

1. Signal activates oligomerisation and hence protein kinase domains. Auto phosphorylation of receptor kinase domain increases kinase activity. Autophos outside domains provide docking sites for proteins with SH2 domains
2. Various proteins including adapter proteins and receptor-associated tyrosine kinases, bind via their SH2 to phosphotyrosine on the receptor

Question 11

How does Grb-2 initiate signalling as an example of modular construction?

Answer 11

The SH2 domain binds to phosphotyrosine residues on an activated receptor, while the SH3 domains bind proline-rich regions on other proteins.
Sos, recruited by the Grb-2 adapter protein, binds to Ras and opens up its nucleotide binding site, allowing GDP to escape and GTP to bind.
Activated Ras binds and activated other proteins

Question 12

How are MAP kinases activated?

Answer 12

Phosphorylation at both Thr and Tyr in a conserved Thr-Gly-Tyr motif on the activation loop

Question 13

What is the structure of p38 MAP kinase?

Answer 13

ATP and phosphorylation of Thr183 and Tyr185 on the activation loop
2 Mg ions are present

Question 14

What are the differences between the active and inactive forms of p38 MAP kinases?

Answer 14

Rearrangement of activation loop to present pThr to C-helix
Rotation of the N-terminal domain
Rearrangement of alpha-carbon
Rearrangement of gly flap