Protein Phosphorylation

Question 1

Phosphorylation may directly alter protein activity by causing: (3 Things)

Answer 1

1. Steric interference between a protein and its ligand
2. Conformational changes: Short and long range
3. Change in binding to another protein

Question 2

Chemical features of protein phosphorylation:

Answer 2

• Phosphate can form 3 or more hydrogen bonds.
• The tetrahedral geometry makes these hydrogen bonds highly directional, allowing for specific interactions with hydrogen‐bond donors
• Phosphate adds negative charges to a protein, so existing electrostatic interactions can be disrupted and new can be formed
• Phosphate forms a stable but not too stable bond (reversible).
• Phosphate is added and removed under control of enzymes and therefore highly ‘regulatable’

Question 3

Example of “Steric interference between a protein and its ligand “

Answer 3

Phosphorylation of Ser113 of Escherichia coli isocitrate dehydrogenase blocks the substrate isocitrate from binding to the active site

Question 4

Example of “Conformational changes: Short and long range”

Answer 4

Insulin Receptor Kinase (IRK)
Activation loop in unphosphorylated IRK blocks binding of substrate.
Activation loops moves upon phosphorylation at 3 Tyrosine (in the activation loop), allowing a conformational change that allows a substrate binding to the kinase.

Question 5

Example of “Change in binding to another protein”

Answer 5

FHA Domain: Fork Head Associated Domain

• Modular domains within a variety of multidomain proteins in eukaryotes
• 80-100 Amino acids
• Most FHA domains recognise phosphoThr with specificity provided by residues C-terminal to the phosphoThr residue, particularly the +3 position.

Question 6

Two ATPs are hydrolyzed in every kinase/phosphatase cycle

TRUE OR FALSE

Answer 6

FALSE
One ATP is hydrolyzed in every kinase/phosphatase cycle .
The energy required to “flick the switch”.

Question 7

The amino acid residues that are conserved in protein kinases are vital for function.
They control the position of:

Answer 7

ATP
Mg2+
Substrate Protein
Transfer of electrons (asp in catalytic loop) leading to hydrolysis of ATP and phosphotransfer

Question 8

Protein kinases catalyze phosphate transfer from Mg-ATP (or Mg-GTP) to specific BLANK, BLANK, and BLANK residues in proteins

Answer 8

Serine, Threonine, and Tyrosine (STY)

Question 9

Overview of protein kinases catalysing phosphate transfer from Mg-ATP (or Mg-GTP) to specific STY residues in proteins:

Answer 9

1. The catalytic Asp gains a proton
2. ATP loses a phosphate to become ADP
3. The substrate gains a phosphate group

• Activated protein kinases, the substrate residue (S,T, Y) is close to the γ-phosphate of ATP and to the Asp in the catalytic loop of the protein kinase
• The CO2 on the side chain of the essential catalytic Asp acts as a catalytic base that accepts a proton from the side chain hydroxyl the target residue
• Mg2+ interacts with β and γ phosphates of ATP (enhancing the electronegativity of their oxygen atoms)

Question 10

The Human Kinome:

Answer 10

> 500 human eukaryotic protein kinases
2% of genes
300 AA kinase domains form 2-lobed sequences
Each sequence subfamily has different substrate specificities

Question 11

How are protein kinases controlled? (Many mechanisms, give 4)

Answer 11

1. Phosphorylation of activation loops
2. Other domains in the kinase mediate regulated changes in subcellular location and interactions with activators and substrates
3. Regulated binding of kinase domain with inhibitory peptides, proteins and pseudosubstrate regions
4. Regulated binding of kinase domain with activating proteins

Question 12

Example of “Phosphorylation of activation loops”:

Answer 12

Catalyzed by autophosphorylation (Another kinase of the same type phosphorylates it)

Catalysed by other kinases:

• Tyrosine kinases usually have one or more phospho-tyrosines in the loop
• MAPK kinases have a T[D/E]Y motif that is phosphorylated on both T and Y
• Most other kinases have a phosphorylated threonine within the loop.

Conformations of the activation loops in INACTIVE forms are variable.
Conformations of the activation loops in ACTIVE forms are very similar.

In some inactive forms, the Phe in the DFG (Mg2+-binding) motif at the start of the activation loop swings out (DFG-out conformation) and blocks the ATP-binding site.

Question 13

Example of “Regulated binding of kinase domain with activating proteins”

Answer 13

e.g
1. Binding of its cyclin ligand, which induces a conformational change in the alphaA helix of the Cdk
2.
3.

Question 14

Example of “Regulated binding of kinase domain with inhibitory peptides, proteins and pseudosubstrate regions”

Answer 14

Modulated by chemical messengers such as calcium ions and cyclic AMP

e. g. Protein Kinase A (PKA)
- Catalytic site blocked by a regulatory subunit (R) which has a pseudosubstrate region that blocks the active site.
- The catalytic subunit is liberated by binding of cAMP to the R subunit.
- Activation of PKA also requires phosphorylation of Thr197 in the activation loop and conformational rearrangement

Question 15

Example of “Regulated binding of kinase domain with activating proteins”

Answer 15

e. g
1. Binding of its cyclin ligand, which induces a conformational change in the alphaA helix of the Cdk
2. Phosphorylation of the Cdk (By CAK) in this Cdk-cyclin complex at a particular threonine residue in the Cdk activation loop
3. Dephosphorylation at one or two other tyrosines (depending on the particular Cdk)