Protein Kinases 1

Question 1

Name three ways kinases can fit into signalling pathways.

Answer 1

They can act as receptors, effectors and control cell functions.

Question 2

Describe the structure of a phosphate group.

Answer 2

Central phosphate atom, with three single bonds to O-, and a double bond to O.

Question 3

What enzymes are responsible for addition of phosphate groups?

Answer 3

Kinases.

Question 4

What is the difference between kinases and phosphatases?

Answer 4

Kinases add phosphate groups through a condensation reaction, releasing a water molecule, whereas phosphatases are enzymes responsible for removing phosphate groups.

Question 5

Which amino acid residues are the primary focus of phosphorylation in eukaryotes?

Answer 5

Serine, threonine and tyrosine.

Question 6

What 5 amino acids can be phosphorylated?

Answer 6

Serine, threonine, tyrosine, histidine and aspartate.

Question 7

What feature do the amino acids that can be phosphorylated have?

Answer 7

All have a hydroxyl group, upon which the phosphate group is added.

Question 8

Aspartate phosphorylation is used in prokaryotes, in which signalling system?

Answer 8

The two component system signalling.

Question 9

What is the source of phosphates for phosphorylation?

Answer 9

Mostly the phosphates come from ATP, however they can also originate from GTP as they both have purine rings and similar systems, despite some differences.

Question 10

Which residues are predominantly phosphorylated in eukaryotes?

Answer 10

Serine and threonine (and tyrosine but to a much lower extent).

Question 11

Why was phospho-tyrosine initially thought to be the most important phosphorylated residue?

Answer 11

Because initially the techniques developed allowed analysis of tyrosine phosphorylation, also because many oncogenes were found to be tyrosine kinases, and so much of the literature focussed on tyrosine kinase phosphorylation.

Question 12

What is the advantage of studying tyrosine signalling despite the tyrosine levels being extremely low?

Answer 12

Because even small changes in tyrosine levels are apparent.

I.e. a greater, more apparent signal gain.

Question 13

What is an archetype?

Answer 13

A very typical example of a certain thing.

Question 14

What is protein kinase A (PKA) and what signal does it respond to?

Answer 14

It is a protein kinase that responds to the second messenger cAMP.

Question 15

Outline the concept behind cAMP signalling.

Answer 15

cAMP is generally present at very low levels within cells, i.e. in relatively short supply, until it is manufactured as part of the signalling mechanism. As it rises, it activates the enzyme PKA.

Question 16

Describe the structure of PKA in the inactive state.

Answer 16

When it is not active, it is ordinarily present in the cytosol in an incapacitated state as a tetramer of 2 regulatory subunits and 2 catalytic subunit bound to 2xADP each (i.e. 2R2C heterotetramer).

Question 17

Outline the activation of PKA.

Answer 17

When cAMP concentrations rise they can bind to the binding sites for cAMP on the regulatory subunits of PKA, which will bidn cAMP in a cooperative fashion (i.e. the binding of the first cAMP increases the affinity at the other binding sites). When cAMP binds to these regulatory subunits, it changes the conformation therefore they can no longer hold onto the catalytic subunits. THe regulatory subunits remain as a dimer, whilst the two catalytic subunits are released as monomers into the cytosol. These free catalytic components are then able to bind ATP and use it to phosphorylate their targets.

Question 18

What is the function of the regulatory subunits of PKA?

Answer 18

They function to hold the enzyme in an inactive conformation whilst they are bound.

Question 19

Describe the overall notable features and structures of PKA.

Answer 19

Small lobe:
-Mostly comprised of beta sheets, involved in ATP binding in the ATP-binding site in the crevice of the middle region.

Large lobe:
-Mostly comprised of alpha helices, and contains the peptide binding site.

A tail.
A phosphorylation site (for the kinase itself).

Question 20

Name the 5 main loops in the PKA structure.

Answer 20

Glycine-rich loop. 
Catalytic loop. 
P+1 loop. 
Mg2+ loop. 
C-Helix (PSTAIRE-like helix).

Question 21

Where is the glycine rich loop located in PKA?

Answer 21

This is a tight loop found between the 1st and 2nd beta strand in the structure.
ca 49-56.

Question 22

Where is the catalytic loop located in PKA?

Answer 22

Between beta strand 6 and 7.

ca 165-172.

Question 23

Where is the P+1 loop located in PKA, and what is it referred to in other kinases?

Answer 23

Found between the 9th beta strand and the 6th (F) alpha helix.
Known as the activation loop in other kinases, as P+1 is specific for PKA.

Question 24

What is the Mg2+ loop, and where is it located?

Answer 24

It is a tight loop that is invariant in all protein kinases.
It has the sequence DFG (aspartate, phenylalanine, glycine).
This sequence occurs regularly and is essential to bind Mg2+.
It is located between amino acids (ca) 184-186 and between beta strands 8 and 9).

Question 25

Why is Mg2+ important for kinase activity?

Answer 25

The kinases won't work without it.

Question 26

Where is the C-helix (PSTAIRE-like helix) located?

Answer 26

Between amino acids (ca) 85-97.

Question 27

Why can PKA be used as an archetype for the structure of other protein kinases?

Answer 27

Because the folds of many other proteins is very similar to that of PKA.

Question 28

Name 4 protein kinases.

Answer 28

Protein kinase A (PKA). Phosphorylase kinase (1PHK). Hck, a close relative of pp60src (2HCK). Casein kinase-1 sigma (1CK1).

Question 29

What process do protein kinases undergo?

Answer 29

They undergo molecular movements in a catalytic cycle of chemical reactions that only goes in one direction.

Question 30

Outline the catalytic cycle of protein kinases.

Answer 30

- ATP binds to the enzyme. - The substrate/peptide binds to the kinase at the same time, resulting in a ternary complex (kinase+ ATP+ structure). - The gamma phosphate is then transferred directly from the ATP to the peptide, giving a phospho-peptide. - This phospho-peptide then leaves the complex as the binding breaks down. - Shortly after, ADP leaves and cycle returns to the start and is repeated.

Question 31

What components are necessary in order for the cycle to repeat?

Answer 31

ADP and substrate.

Question 32

What is the slowest step of the reaction pathway?

Answer 32

The release of ADP, this shows that this step governs the maximum rate of phosphorylation.

Question 33

Outline the interactions of ATP with the protein kinase.

Answer 33

ATP forms bonds to the small lobe of the protein kinase, involving the alpha and beta phosphates, and so do not change throughout the cycle. It also forms bonds with the large lobe, however these are bonds that change as they involved the gamma phosphate which is transferred to the substrate.

Question 34

What bonds does ATP form with the small lobe?

Answer 34

ATP forms bonds that don't change with the small loop: - Glycine rich loop. - Beta-strand 2. - Beta-strand 3.

Question 35

Describe the bond formed by ATP to the glycine rich loop.

Answer 35

Backbone amides of F54 and G55 make H-bonds to ATP beta phosphate. Therefore, the small lobe glues itself to parts of ATP that don't change in the reaction cycle. Very mobile.

Question 36

Describe the bond of ATP to the beta-strand 2.

Answer 36

There is a V57 hydrophobic interaction with the purine ring of ATP. The beta strand attaches itself to another invariant part of ATP that does not change in the catalytic cycle.

Question 37

Describe the bond of ATP to the beta-strand 3.

Answer 37

K27 ion (lysine in the PKA sequence) W

Question 38

What bonds are important in the large lobe for the catalytic cycle?

Answer 38

Bonds with: - Catalytic loop (beta 6-7). - Mg2+ binding loop (beta 8-9). - Activation loop. - P+1 loop. - F-helix.

Question 39

Describe the importance of the bond formed by the catalytic loop of the large lobe.

Answer 39

The K168 ion pairs to the gamma phosphate of ATP.

Question 40

Describe the importance of the bond formed by the Mg2+ loop of the large lobe.

Answer 40

D184 ligand binds to the Mg2+, which binds tightly to ATP alpha and beta phosphates.

Question 41

What is the role of the P+1 loop in the catalytic cycle?

Answer 41

This loop plays a key role in docking the substrates to the kinase.

Question 42

What is the importance of the F-helix in the catalytic cycle?

Answer 42

It underpins the essential stable core of the enzyme and contributes to the catalytic competence over long distances.

Question 43

Describe the organisation of the protein kinase prior to the transfer of the phosphate group to the substrate.

Answer 43

Prior to the transfer of the phosphate from Pi-> Peptide... the small and large lobes are held closely together, as they are each making strong contacts with teh ATP. I.e. the ATP acts like a glue for these two lobes.

Question 44

Which phosphate is transferred from the ATP to the substrate?

Answer 44

Gamma phosphate.

Question 45

What happens in the small lobe once the gamma phosphate has been transferred to the peptide?

Answer 45

The bonds made between the small lobe and the ATP remain intact as they concern the purine ring, the alpha and the beta phosphates, which all remain unchanged in the hydrolysis of ATP-> ADP.

Question 46

What happens in the large lobe once the gamma phosphate has been transferred to the peptide?

Answer 46

The significant interactions that were formed between the large lobe and the gamma phosphate have been transferred to the peptide, along with the phosphate group.

Question 47

What happens as a result of this transfer of bonds?

Answer 47

The phospho-peptide is ejected, resulting in subtle changes in the organisation of the active sites as you progress through the catalytic cycle. The re-organisation is what aids the ejection of the newly formed peptide and ADP.

Question 48

What happens when the cycle restarts and a new ATP and peptide molecule bind to the kinase?

Answer 48

The lobes will close together again and the cycle will repeat itself.

Question 49

What are the two distinct roles of purine nucleotides in the catalytic cycle of the protein kinases?

Answer 49

1) To serve as a donor of Pi during the phosphorylation reactions, from the gamma phosphate of ATP. 2) To contribute to adoption of a particular conformation by the kinase protein/ kinase domain (because when ADP/ ATP are bound by the kinase, conformational changes are induced by stabilising teh hydrophobic catalytic spine).

Question 50

Where are polar amino acids normally found within a protein, and give examples?

Answer 50

I.e. polar amino acids are almost always found/ enriched on the surface of the protein i.e. serine, threonine and tyrosine.

Question 51

In what way are kinases highly selective?

Answer 51

Because not all hydroxyl-containing amino acids will be phosphorylated in cells, even during cell signalling... i.e. it is reproducibly the same ones that accept a phosphate.

Question 52

How does the selectivity of the kinases arise?

Answer 52

Physical accessibility. Cryptic sites. Consensus sequences.

Question 53

How does physical accessibility influence kinase selectivity?

Answer 53

Loops and turns are preferred.

Question 54

How do cryptic sites influence kinase selectivity?

Answer 54

I.e. meaning that some change must first occur in the protein structure in order to reveal the phosphorylation site. I.e. not all phosphorylation sites are immediately accessible, and could be hidden until another trigger exposes them so that the kinase can see them.

Question 55

How do consensus sequences influence kinase selectivity?

Answer 55

It is not just the amino acid to be phosphorylated which is recognised by the kinase... it recognises the flanking amino acid sequence too, and exhibits selectivity towards the flanking amino acid sequence.

Question 56

What is meant by a consensus sequence?

Answer 56

The sequence of amino acids flanking the amino acid to be phosphorylated... will be conserved... like a key to fit a lock on the kinase. There is a tight code selection to see exactly which proteins fit into the site to be phosphorylated. So the kinase not only recognises a specific amino acid... but it is also cognate to the surrounding consensus sequence.

Question 57

What methods allow you to discover the consensus sequences?

Answer 57

Multiple sequencing alignment. | Degenerate peptide libraries.

Question 58

What is the concept behind multiple sequence alignment?

Answer 58

Used amongst known targets of kinases, where the frequency of the occurrence of one amino acid/ type of amino acid can help to discover a pattern in an apparent specificity. This pattern can then be used to identify new potential substrates. Can then search for the sequences that will best fit a kinase, via computers.

Question 59

Briefly outline the process of multiple sequencing alignment/

Answer 59

I.e. say you have a protein kinase (PKA) and you want to know what the proteins that it will phosphorylate are... can take say 5 substrates that that protein kinase phosphorylates, and look to see what they have in common, i.e. look at the flanking sequence around the amino acid that is phosphorylated... can put together a consensus sequence of the different amino acids/ types of amino acids that are required. Once you have the consensus sequence, you can search the protein database to see if any other proteins have that sequence... may be another potential substrate for the kinase.

Question 60

What is the benefit of multiple sequence alignment?

Answer 60

It doesn't necessarily prove that if a certain kinase has a similar sequence that it can be phosphorylated by the kinase, however it makes the process more efficient as it gives scientists greater predictive power.

Question 61

What is the disadvantage of the multiple sequencing alignment technique?

Answer 61

That not all targets of a kinase may be known, and so it is biased towards the known targets.

Question 62

Outline the main concept behind degenerate peptide libraries.

Answer 62

This is where the kinase is allowed to react with a combinatorial library of substrates containing billions of random combinations of amino acids. The phosphorylated forms are extracted, and you can search for enrichment of particular amino acids in particular positions. Essentially, you synthesise billions of random peptides, with different sequences.

Question 63

Outline the process of degenerate peptide libraries.

Answer 63

So you will synthesise billions of random peptides, each with different sequences. You can specify the middle amino acid to be fixed i.e. serine... tehn randomise the flanking sequences. Once you have your peptide library, you can add the kinase that you are interest in, add the ATP and Mg2+, then allow it to carry out its activity and phosphorylate the particular sequences. Then you stop the enzyme reaction by removing the magnesium to stop it, and can take all of these peptides to enrich the ones that have been phosphorylated by the kinase. Usually, you can use an affinity step i.e. beads or something covered in a metal ion that will interact with the phosphate. Can now suck out all the phosphorylated peptides and throw away the rest. You now have a sub-population of the random peptide library that you had, and can pass them into the amino acid sequencer to see which amino acids are enriched at the particular positions, to reveal the preferred amino acid sequence of the kinase.

Question 64

Why are good consensus sequences important?

Answer 64

Because the more mutually agreeable interactions there are between the two surfaces, i.e. opposites, positives vs negatives etc. the longer these will stay associated, giving a greater likelihood of a successful stochastic reaction process.