Topic 17: Post-translational modifications: Phosphorylation

Question 1

What is the fundamental definition of protein phosphorylation?

Answer 1

It is the reversible, covalent modification of a protein by attaching a phosphate group from ATP to the side chains of specific amino acids.

Question 2

What are the respective roles of a kinase and a phosphatase in the phosphorylation cycle, often called the ‘writer/eraser’ system?

Answer 2

A kinase (‘writer’) adds a phosphate group to a substrate, while a phosphatase (‘eraser’) removes it.

Question 3

Approximately how many genes for kinases and protein phosphatases are encoded in the human genome?

Answer 3

There are approximately 500 kinase genes and 140 protein phosphatase genes.

Question 4

Protein kinases are broadly divided into two main classes based on their substrate specificity. What are these two classes?

Answer 4

Serine/threonine (Ser/Thr) kinases and Tyrosine (Tyr) kinases.

Question 5

Which amino acid residues are phosphorylated by Ser/Thr kinases?

Answer 5

They phosphorylate the hydroxyl group on the side chains of serine and threonine residues.

Question 6

What is the approximate distribution of Ser/Thr kinases versus Tyr kinases in the human genome?

Answer 6

There are approximately 400 Ser/Thr kinases and 100 Tyr kinases.

Question 7

What is the evolutionary significance of tyrosine kinases in relation to organism complexity?

Answer 7

Tyrosine kinases are found almost exclusively in metazoans (multicellular animals), suggesting their signalling role emerged around the time multicellularity evolved.

Question 8

Describe the conserved core structure of a protein kinase.

Answer 8

It consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe), with the active site located in the cleft between them.

Question 9

What two key molecules bind within the active site cleft of a protein kinase?

Answer 9

The active site binds both ATP (the phosphate donor) and the peptide substrate (the phosphate acceptor).

Question 10

What is the essential role of a divalent cation, such as Mg
2+
, in the catalytic mechanism of a kinase?

Answer 10

It is required to coordinate the negatively charged phosphate groups of ATP, holding it in the correct orientation for hydrolysis and phosphate transfer.

Question 11

The catalytic reaction of a kinase involves a _____ attack on the ATP γ-phosphate by the hydroxyl group of the substrate’s amino acid.

Answer 11

nucleophilic

Question 12

Which specific phosphate group from ATP is transferred to the substrate during phosphorylation?

Answer 12

The terminal, or gamma (γ), phosphate group is transferred.

Question 13

What is the function of the ‘activation loop’ within the C-lobe of a kinase?

Answer 13

It provides a platform for the peptide substrate to bind and is often phosphorylated itself to stabilise an active, open conformation.

Question 14

In the catalytic mechanism of protein kinase A (PKA), which residue acts as a general base to activate the substrate’s hydroxyl group for nucleophilic attack?

Answer 14

Aspartate 166 (Asp166), located on the catalytic loop, acts as the general base.

Question 15

What are the two products of the kinase-catalysed phosphorylation reaction?

Answer 15

The products are the phosphorylated protein and MgADP.

Question 16

How do many modern anti-cancer drugs, such as Imatinib (Gleevec) and Vemurafenib, inhibit kinase activity?

Answer 16

They are designed to mimic ATP and act as competitive inhibitors, binding in the ATP-binding pocket of the kinase active site to block its function.

Question 17

The BRAF (V600E) mutation is common in melanoma. How does this specific mutation lead to a constitutively active kinase?

Answer 17

The mutation substitutes a valine with a negatively charged glutamic acid, which mimics the negative charge of a phosphate group, locking the kinase in a permanently ‘on’ state.

Question 18

Name one of the four main mechanisms by which a kinase achieves specificity for its target substrate.

Answer 18

1) Recognising sequence motifs around the phosphorylation site, 2) Using docking sites away from the active site, 3) Containing modular domains to recruit substrates, or 4) Using scaffold/adaptor proteins.

Question 19

Which group of kinases, including MEK1 and MEK2, is unusual for its ability to phosphorylate both tyrosine and threonine residues?

Answer 19

Dual-specificity kinases.

Question 20

Contrast the relative abundance of pSer/Thr phosphatases and pTyr phosphatases in the human genome.

Answer 20

There are far more tyrosine phosphatases (~107) than serine/threonine phosphatases (~28), which is the opposite of the distribution seen in kinases.

Question 21

What is the key feature of the active site of serine/threonine phosphatases?

Answer 21

They are metalloenzymes with a bimetal active site (typically containing ions like Fe
3+
, Zn
2+
, or Mn
2+
) that coordinates the substrate and activates a water molecule.

Question 22

What molecule performs the nucleophilic attack to dephosphorylate the substrate in the active site of a Ser/Thr phosphatase?

Answer 22

An activated water molecule performs the nucleophilic attack, transferring the phosphate group to water.

Question 23

What is the key catalytic residue in the active site of most Class I tyrosine phosphatases?

Answer 23

An active-site cysteine (Cys) residue acts as the initial nucleophile.

Question 24

Describe the two-step reaction mechanism of a Cys-based tyrosine phosphatase.

Answer 24

First, the active-site cysteine attacks the phosphate, forming a covalent thiophosphate intermediate and releasing the dephosphorylated protein. Second, water attacks this intermediate to release the free phosphate.

Question 25

What defines a Dual-Specificity Phosphatase (DUSP)?

Answer 25

A DUSP is a phosphatase that can dephosphorylate phospho-serine (pSer), phospho-threonine (pThr), and phospho-tyrosine (pTyr) residues.

Question 26

In the MAPK pathway, RAF phosphorylates MEK, and MEK subsequently phosphorylates ERK. What is unusual about the phosphorylation of ERK by MEK?

Answer 26

MEK, a dual-specificity kinase, phosphorylates ERK on both a threonine and a tyrosine residue.

Question 27

How do the DUSPs, DUSP5 and DUSP6, regulate the localisation and activity of ERK?

Answer 27

DUSP6 sequesters and dephosphorylates ERK in the cytoplasm, while DUSP5 performs the same function in the nucleus.

Question 28

Some DUSPs can act on non-protein substrates. What is the classic example of such a DUSP and its lipid substrate?

Answer 28

PTEN is a DUSP that acts as a lipid phosphatase, dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ​ ) to produce PIP 2 ​ .

Question 29

The kinase _____ adds a phosphate to PIP 2 ​ to create the signalling molecule PIP 3 ​ .

Answer 29

PI3 kinase (PI3K)

Question 30

What is the primary function of PIP 3 ​ at the plasma membrane after it is generated by PI3K?

Answer 30

PIP 3 ​ acts as a docking site, recruiting signalling proteins like Akt (Protein Kinase B) and PDK1 to the membrane via their PH domains.

Question 31

What signalling pathway, crucial for cell growth and survival, is negatively regulated by the lipid phosphatase PTEN?

Answer 31

The Akt (or Protein Kinase B) pathway.

Question 32

In the 'writer/eraser' analogy, the kinase is the 'writer' and the _____ is the 'eraser'.

Answer 32

phosphatase

Question 33

Given that PTEN switches off a pro-growth and pro-survival pathway, it is functionally classified as a _____.

Answer 33

tumour suppressor

Question 34

What provides the platform for the peptide substrate to bind in the active site of most kinases?

Answer 34

The activation loop, which is part of the C-lobe

Question 35

Why do tyrosine kinases require a different catalytic cleft shape compared to serine/threonine kinases?

Answer 35

They need to accommodate the bulky aromatic ring of the tyrosine side chain, which is much larger than the side chains of serine or threonine.

Question 36

What is the mechanism of action for the leukaemia drug Imatinib (Gleevec)?

Answer 36

It inhibits specific tyrosine kinases, such as BCR-ABL, by blocking their ATP-binding site.

Question 37

A kinase that uses an accessory protein to bind both the kinase and its substrate is utilising a _____ or _____ protein.

Answer 37

scaffold, adaptor

Question 38

Contrast the catalytic mechanisms of Ser/Thr phosphatases and Cys-based Tyr phosphatases regarding the initial nucleophile.

Answer 38

Ser/Thr phosphatases use an activated water molecule as the nucleophile, whereas Cys-based Tyr phosphatases use an active-site cysteine residue as the initial nucleophile.

Question 39

What happens to ERK's cellular location after it is phosphorylated by MEK?

Answer 39

Phosphorylation causes it to dissociate from MEK in the cytoplasm, leading to its accumulation in the nucleus.

Question 40

Why is a dual-specificity phosphatase required to fully inactivate ERK?

Answer 40

Because ERK is activated by phosphorylation on both a threonine and a tyrosine residue, a DUSP is needed to remove both phosphate groups.

Question 41

In many cancers, PTEN is mutated or lost. What is the direct biochemical consequence of this loss at the cell membrane?

Answer 41

The loss of PTEN leads to an accumulation of PIP 3 ​ at the cell membrane, as it cannot be dephosphorylated back to PIP 2 ​ .

Question 42

The accumulation of PIP 3 ​ due to PTEN loss leads to the hyperactivation of which downstream kinase that promotes cell survival and growth?

Answer 42

Akt (Protein Kinase B).

Question 43

Proteins like Akt and PDK1 are recruited to the membrane by binding to PIP 3 ​ via their _____ domains.

Answer 43

pleckstrin homology (PH)

Question 44

How does DUSP6 regulate ERK in the cytoplasm?

Answer 44

It competes with MEK for ERK binding and causes sequestration of dephosphorylated ERK in the cytoplasm.

Question 45

Which two key catalytic residues in a kinase active site help coordinate the ATP molecule?

Answer 45

Lysine 72 (Lys72) from the N-lobe and Aspartate 184 (Asp184) from the C-lobe.

Question 46

The dysregulation of kinases and phosphatases are important drivers in diseases such as _____.

Answer 46

cancer

Question 47

Kinases that have docking sites away from the active site that recognize docking-motif peptides are typically which class of kinase?

Answer 47

Serine/threonine (Ser/Thr) kinases.

Question 48

What is the name of the covalent intermediate formed during the catalytic cycle of a Cys-based tyrosine phosphatase?

Answer 48

A thiophosphate intermediate.

Question 49

What is the function of DUSP5?

Answer 49

DUSP5 is a nuclear-targeted phosphatase that sequesters dephosphorylated ERK in the nucleus.