Block A Lecture 2 - Protein Kinases

Question 1

What are the 3 most well studied / known kinases?

Answer 1

Serine / Threonine kinases (~99%) and tyrosine kinases
(Slide 4)

Question 2

How does glucagon result in the activation of protein kinase A (PKA) and what does this do after its activation?

Answer 2

1. Glucagon binds to its GPCRs on the surface of target cells (usually liver cells).
2. This activates the G protein on the receptor, which then goes on to activate adenylate cyclase, which is an enzyme in the cell membrane.
3. Adenylate cyclase converts ATP into cyclic AMP (cAMP) which acts as a 2nd messenger inside the cell.
4. cAMP binds to the regulatory subunits of protein kinase A (PKA) resulting in it becoming activated and being able to phosphorylate targets.
5. PKA then phosphorylates glycogen synthase a into glycogen synthase b, turning it off and therefore inhibiting glycogen synthesis.
6. At the same time, PKA phosphorylates glycogen phosphorylase b into glycogen phosphorylase a, promoting glycogen degradation.

(Slide 8)

Question 3

How do protein kinases “amplify” a signal?

Answer 3

1 molecule of the kinase results in multiple of whatever it phosphorylates being activated (e.g 1 protein kinase A results in 10 phosphorylase kinase being activated which results in 1000 glycogen molecules being degraded etc.)
(Slide 9)

Question 4

What 4 subunits is protein kinase A made up of?

Answer 4

It’s a R2C2 complex, made up of 2 R (regulatory) subunits and 2 C (catalytic) subunits.

(Slide 12)

Question 5

What mechanism does cAMP use to activate protein kinase A?

Answer 5

It binds to the R subunit of protein kinase A, resulting in the R2C2 complex dissociating, activating the C subunits (as the active site is revealed) and making them capable of phosphorylating targets

(Slides 12 and 22)

Question 6

Sequence alignment and biochemical analysis has shown that kinases adopt similar structures in terms of their geometry and active site shape, showing that their basic catalytic mechanism is conserved. What does this mean?

Answer 6

That almost all kinases work in the exact same way, so learning the general catalytic mechanism of one kinase means that you will essentially know the catalytic mechanism of all kinases
(Slide 14)

Question 7

What is the general catalytic mechanism that most kinases use?

Answer 7

1. ATP binds to the active site of the kinase.
2. The substrate also binds at the active site.
3. The γ-phosphate is transferred from ATP to a serine/threonine / tyrosine kinase.
4. The substrate is released from the kinase.
5. ADP is released from the active site.

(Slide 15)

Question 8

What is one example of a conserved sequence motif that most kinases have?

Answer 8

A “DFG” motive present at the active site.
(Slide 19)

Question 9

What are 3 ways that kinases are activated by unmasking of the active site?

Answer 9

cAMP binding
Use of an activation loop
“Pseudo-substrate” domains
(Slide 23)

Question 10

How does an activation loop result in a kinase being inactive in its default state?

Answer 10

As it physically blocks access to the active site, preventing any substrate from being able to bind to it
(Slide 24)

Question 11

How does phosphorylation of an activation loop result in a protein kinase being activated?

Answer 11

As the activation loop being phosphorylated results in a conformational change, moving the loop out of the way of the active site, allowing a substrate to bind
(Slide 25)

Question 12

What is a pseudosubstrate region?

Answer 12

A small sequence of amino acids which mimic a substrate and bind the substrate-binding cavity in the catalytic domain of a protein kinase
(Slide 34)

Question 13

Why does a pseudosubstrate region binding to the substrate-binding cavity of a kinase not activate the kinase, like when a real substrate binds to this region?

Answer 13

As a pseudosubstrate region lacks serine or threonine phospho-acceptor residues, thus keeping the enzyme inactive
(Slide 34)

Question 14

What results in the release of the pseudosubstrate from the catalytic site (and therefore the activation of the kinase / enzyme)?

Answer 14

Interaction with the (cell) membrane
(Slide 36)

Question 15

In the case of protein kinase C (PKC) what is the “interaction with the membrane” which results in the release of the pseudosubstrate from the catalytic site?

Answer 15

DAG binding to the C1 domain
(Slide 36)

Question 16

What does DAG binding to the C1 domain being the interaction which results in the release of the pseudosubstrate domain show about the activation mechanism of protein kinase C?

Answer 16

This interaction can only happen if both calcium and DAG are present; meaning that the activation is precisely controlled and unlikely to happen by mistake
(Slide 36)

Question 17

Since the active site structure, geology and catalytic mechanisms of protein kinases are mostly conserved, what can be said about the sequences that their target proteins have at their site of phosphorylation?

Answer 17

That sequences at the site of phosphorylation on target proteins are similar to each other as this interacts with the kinase active site (which as previously mentioned, active site structures are highly conserved between kinases)
(Slide 37)

Question 18

In addition to changing the conformation of its target, what are 4 other affects that phosphorylation can have on a target protein?

Answer 18

Change its localisation
Change its interactions (such as increasing / decreasing affinity for a target)
Change its half life (made it degrade more or less quickly)
Change its sensitivity to a signal
(Slide 41)