Signal transduction and the Cell cycle (L11)

Question 1

What are the functions of protein kinases?

Answer 1

Bind ATP and protein substrates and phosphorylates target proteins

Question 2

What is the structure of a protein kinase?

Answer 2

Bi-lobal fold
N-terminal domain mainly beta-sheet
C-terminal domain mostly alpha-helical
Two domains linked by a flexible hinge (usually open in inactive conformation and closed in active conformation)
ATP binding site located in the deep cleft between the two lobes

Question 3

How are protein kinases internally conserved?

Answer 3

1. Gly flap is flexible and folds over the ATP site, orients ATP phosphates in active conformation. GXGXXG motif. Backbone NH binds to ATP phosphate
2. The C-helix where a conserved glutamate E interacts with a conserved lysine in strand 3 which itself interacts with the ATP phosphates
3. Catalytic loop (DXKXXN) which is involved in the nucleophillic attack of target amino acid OH by conserved Asp. Transfer of gamma phosphate from ATP
4. The activation loop which undergoes large structural rearrangement on activation. Contains phosphorylation and other sites involved in substrate recognition

Question 4

What diseases are linked to excessive G protein signalling?

Answer 4

Cholera
Cancer if pituitary and thyroid
Cancer of adrenal and ovary
Essential hypertension

Question 5

What diseases are linked to deficient G protein signalling?

Answer 5

Night blindness
Pseudohypoparathyroidism type 1b
Pertussis (Whooping cough)

Question 6

How do G-proteins initiate PKA signalling?

Answer 6

1. Activation initiated by G-protein linked receptor at cell surface
2. Incoming signal at cell surface picked up by G-protein linked receptor. Intracellular membrane anchored G-protein responds
3. G-protein activated by receptor and the active alpha subunit dissociates
4. Activated G-protein alpha subunit activates adenyl(yl/ate) cyclase which then produced cyclic AMP
5. cAMP activates PKA by inducing PKA to dissociate from its regulatory protein
6. Activated PKA migrates to the nucleus where it stimulates the expression of specific genes through phosphorylation and activation of the transcriptional activator CREB

Question 7

In what forms is G-alpha inactive and active?

Answer 7

Active in the GTP-bound form

Inactive in the GDP-bound form

Question 8

How does the inactive form of G-alpha switch to the inactive form?

Answer 8

In the active form the three ‘switches’ interact directly or indirectly with the GTP gamma phosphate. The molecular surface is exposed by dissociation of the beta and gamma subunits. The switch regions are coordinated by a magnesium ion and the regions form hydrogen bonds to the additional phosphate of ATP

Question 9

What is adenylate cyclase?

Answer 9

Integral membrane protein

Question 10

How is serine/threonine kinase activated?

Answer 10

Activated by cAMP and phosphorylation of Thr197 on activation loop. Phosphotransfer from the bound ATP to a Ser/Thr on the substrate