Languages of signal transduction- phosphorylation and localisation

Question 1

What is phosphorylation

Answer 1

1. Addition of phosphate group to proteins is a frequently used mechanism to regulate protein activity – a key mechanism for altering structure and function.
2. Added by protein kinases -> ‘writer enzyme’
3. Removed by protein phosphatases -> ‘eraser enzyme’

Question 2

Why is phosphorylation so commonly used for biological regulation?

Answer 2

1. Phosphorylation allows for specific, tightly regulated, and flexible control of protein function.
2. Provides excellent points of kinetic control:
3. Although hydrolysis of ATP or of a phosphorylated residue may be highly energetically favourable, these reactions are, by themselves, kinetically unfavourable—hydrolysis is extremely slow in the absence of a catalyst (kinase / phosphatase).

Question 3

What are the most prevalent kinases

Answer 3

1. The most prevalent kinases in eukaryotic cell communication are Ser/Thr protein kinases and Tyr protein kinases (plus some lipid and dual-specificity kinases).
2. Likewise, there exist Ser/Thr and Tyr phosphatases (as well as dual-specificity phosphatases).

Question 4

Describe the phosphate group

Answer 4

1. negatively charged (-2) –
2. small size but substantial chemical change:
3. e.g. the phosphate group is attracted to positively charged groups nearby and can disrupt hydrophobic interactions or repel negatively charged groups nearby.

Question 5

How can phosphorylation alter secondary structures

Answer 5

1. Local disruption

2. Local ordering

Question 6

Describe local disruption

Answer 6

1. phosphorylation can lead to dramatic steric or electrostatic effects.
2. Due to repulsion of another nearby negative charge, or disruption of a hydrogen bond made by the non-phosphorylated form of the side chain.
3. The resulting conformational changes could, for example, move active-site residues of an enzyme out of position, resulting in a loss of activity.

Question 7

Describe local ordering

Answer 7

1. phosphorylation leads to formation of new structures.

2. Phosphate group participates in new interactions with nearby positively charged moieties.

Question 8

How can phosphorylation can alter tertiary and quaternary structures

Answer 8

1. Phosphorylation can also have longer-range effects on tertiary and quaternary structure.
2. Long range disruption
3. Long range ordering

Question 9

Describe long range disruption

Answer 9

1. phosphorylation can prevent the binding of a protein to a partner molecule or to another domain of the same protein.
2. Addition of negatively charged phosphate group to binding surface may sterically and electrostatically block ligand or substrate binding

Question 10

Describe long range ordering

Answer 10

1. phosphorylation can also promote new long-range intramolecular and intermolecular interactions.
2. Signalling proteins often contain protein interaction domains, such as SH2 domains, that specifically recognize phosphorylated amino acid motifs.

Question 11

What makes intracellular signals precise and specific?

Answer 11

1. Specificity of the interaction between signalling molecules
2. High threshold for signal activation is maintained by constitutively active ‘eraser’ enzymes
3. A protein kinase covalently adds a phosphate from ATP to the signalling protein, and a protein phosphatase removes the phosphate.
4. Many signalling proteins are activated by dephosphorylation rather than by phosphorylation.
5. A GTP-binding protein is induced to exchange its bound GDP for GTP, which activates the protein; the protein then inactivates itself by hydrolyzing its bound GTP to GDP.
6. Backup mechanisms
7. Signal integration- E.g needs to be activated by two different signalling cascades

Question 12

Describe backup mechanisms

Answer 12

1. many signals employ two parallel pathways to activate a single common downstream target protein.
2. Ensures if one malfunctions the other is still active to transduce the signal

Question 13

Describe structure of SRC family kinases

Answer 13

1. SRC family kinases are molecular signal integration devices
2. Multidomain structure- 3 domains
3. Sh2
4. SH3
5. Kinase domain
6. Connected by flexible linker domains

Question 14

Describe mechanism of activating SRC family kinase

Answer 14

1. Inactive state
2. Active site is blocked by activation loop- Compact
3. SH2 binds to phosphorylated tyrosine domain on terminal string
4. SH2 also binds to linker structure between Kinase and SH2
5. If phosphate is removed- Loosens structure
6. SH3 can now bind to activating ligand – must have unfolded stretch of polypeptide that contains multiple proline residues so SH2 can also bind
7. Tyrosine loop structure is blocking substrate attaching
8. Kinase can now phosphorylate tyrosine to self-activate
9. Important in specialised cell matrix adherence junctions enabling cells to crawl and move along substrates

Question 15

What determines kinase specificity

Answer 15

1. Many different mechanisms control exactly which Ser, Thr or Tyr residues get phosphorylated by an active kinase:
2. active site complementarity (amino acids immediately surrounding active site).
3. docking sites elsewhere in the kinase domain.
4. modular binding domains appended to the kinase domain (like SH2/SH3 etc.) to recruit target substrates
5. scaffolds/adaptors different protein stabilises complex between kinase and substrate (like Cyclin/CDKs)

Question 16

How can phosphorylation work with ubiquitination

Answer 16

1. Phosphorylation can work in concert with ubiquitination to control protein degradation
2. When protein is phosphorylated, the phosphate serves as a marker for recognition site of ubiquitin ligase complex
3. Ubiquitin complex then attaches a ubiquitin tail to the protein
4. Signal that marks the protein to targeting to proteosome where it becomes proteolytically degraded

Question 17

Why and how is localisation used to control signalling

Answer 17

1. Proteins are not evenly distributed throughout the cell - this is essential for normal cellular function.
2. Changes in localisation (i.e. local concentration) hugely influence probability of interaction between molecules.
3. Enzymatic or binding reactions are proportional to concentration of reactants
4. Co-localisation of reacting components drives reactions much more efficiently- E.g. two signalling proteins

Question 18

What are the most common cellular translocations involved in signalling

Answer 18

1. Movement of proteins to and from the nucleus

2. Movement of proteins to and from cell membranes

Question 19

Describe movement of proteins to and from the nucleus

Answer 19

1. Genomic DNA and other chromatin constituents such as histones are found exclusively in the nucleus.
2. Access to nucleus is essential for the activity of proteins that act on chromatin, such as chromatin modifiers (post translational modifier of chromatin) and transcription factors.
3. Elaborate mechanisms that regulate nuclear import and export – regulated by signalling.

Question 20

Describe signalling for movement of proteins to and from cell membranes

Answer 20

1. Many signalling proteins and their substrates are exclusively found on membranes.
2. Including transmembrane proteins and proteins containing covalently attached lipid groups, as well as lipids themselves, which are frequent targets of modification in signalling.
3. Confining molecules to the membrane effectively increases the local concentrations.
4. Higher concentration at membrane increases probability that adaptor proteins will bind to transmembrane receptor and increases efficiency of the signal that is being transduced into the cell

Question 21

Describe formation of signalling complexes at the plasma membrane

Answer 21

1. E.g. insulin receptor signalling
2. Activation of insulin receptor triggers formation of signalling complex by recruitment adaptor proteins to the plasma membrane
3. Enabling activation of ras/raf map kinase cascade