Block D Lecture 1 - Protein Kinases in Signal Transduction

Question 1

How has scientists understanding of signal transduction changed over time?

Answer 1

Scientists used to think transduction consisted of a linear signalling pathway with few components which was spatially separated.

Now they realised it consists of a branched signalling pathway (a network) comprised of many components and that the signal isn’t on or off, it is a gradient controlled by phosphorylation.

(Slide 5)

Question 2

What is the definition of a kinase?

Answer 2

An enzyme which transfer the γ-phosphate residue from ATP to substrates

(Slide 7)

Question 3

How are phosphomonoesters formed?

Answer 3

Via phosphorylation of an alcohol and / or phenol groups of the protein substrate

(Slide 7)

Question 4

What does phosphorylation cause?

Answer 4

A change in the properties of the substrate, such as modifying their activity, affinity for binding a different protein or changing their localisation

(Slide 7)

Question 5

What are 3 examples of groups of eukaryotic protein kinases?

Answer 5

Answers Include:

Tyrosine Kinases (TK)

Tyrosine kinase-like kinases (TKL)

Casein kinases (CK1)

Homologues to sterile kinases from yeast (STE)

CMGC - group which includes cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSKs) and cyclin dependent kinase-related kinases (CDK-related kinases)

Calcium calmodulin-dependent kinases (CAMK)

AGC kinases, includes PKA, PKC and PKG

Atypical kinases (AKs)

(Slide 8)

Question 6

What are pseudokinases?

Answer 6

Kinases which consist of a kinase domain and a pseudokinase domain and have lost 1 or more catalytically active resides, meaning they have no catalytic activity (can’t catalyse phosphorylation)

However, they still have a function and act as regulators of active kinases through interaction with their pseudokinase domain

(Slide 11)

Question 7

What is an example of a pseudokinase?

Answer 7

Answers Include:

JAKs 1 - 3b

Tyk2b

GCN2-b

(Slide 11)

Question 8

What is the general structure of the kinase domain a eukaryotic protein kinase?

Answer 8

They contain 12 kinase subdomains and all kinases have 9 conserved β-strands and 6 conserved α-helices

(Slide 12)

Question 9

What was the first protein kinase scientists found the full structure of?

Answer 9

Protein kinase A (PKA), in 1991

(Slide 13)

Question 10

What domains does the p38 kinase have and what are these comprised of?

Answer 10

It is comprised of 2 domains, a smaller N-terminal domain composed on 7 β-strands and a special α-helix (αC) and a larger C-terminal domain which is predominantly α-helical

Question 11

What are the 7 main features of the p38 kinase, and what are their functions?

Answer 11

Lysine-glutamate salt bridge: Stabilises the N-terminal domain which is important for positioning ATP

ATP-binding site: where ATP binds, located in a cleft (gap) between the 2 domains

P-loop - located between the β1 and β2 strands and recognises and binds the phosphate groups of ATP using the GXGXXG motif.

Catalytic loop - has a HRD motif which helps transfer the phosphate group from ATP to the substrate

Activation loop - involved in substrate binding, with phosphorylation of this activating the kinase and allowing the substrate to bind

DFG motif - a highly conserved motif of all kinases which is at the beginning of the activation loop and helps co-ordinate MG2+ for ATP.

Substrate binding site - located at the outer rim of the nucleotide-binding pocket close to the γ-phosphate of ATP, with the activation loop being involved in the binding of the substrate

(Slide 14)

Question 12

What does the limited number of protein kinases in a cell result in?

Answer 12

The same kinase participating in more than one signalling cascade

(Slide 17)

Question 13

What are 3 ways in which protein kinase specificity regulated?

Answer 13

Docking interactions

Scaffold proteins

Mutual inhibition

(Slide 17)

Question 14

What are docking interactions and how do they regulate protein kinase specificity?

Answer 14

Kinases have short amino acid sequences or domains which recognise and bind to complimentary motifs on substrate proteins.

This increases specificity by guiding the kinase to the right protein

(Slide 17)

Question 15

What are scaffold proteins and how do they regulate protein kinase specificity?

Answer 15

Scaffold proteins are proteins which organise multiple enzymes in a pathway into a complex.

It ensures kinases can be localised to specific parts of the cells, improving efficiency, and the pathway can be triggered quicker.

Also ensures signalling is compartmentalised, so that the same kinase can act on different pathways and produce different effects depending on where in the cell it is anchored.

(Slide 17)

Question 16

What is mutual inhibition, and how does it regulate protein kinase specificity?

Answer 16

A situation where 2 pathways or proteins inhibit each other, creating a “switch-like” behaviour, where if one is active the other is off.

This prevents a kinase which can activate multiple pathways from triggering everything at once and ensures exclusive pathway activation so the cell commits to one specific outcome, as sometimes these pathways can produce opposing effects.

(Slide 17)

Question 17

How are protein kinases “molecular switches”?

Answer 17

As their confirmation varies between 2 extremes, they are either on and express maximal activity or are off and experience minimal activity.

(Slide 18)

Question 18

What are 4 examples of way protein kinase activation can be regulated?

Answer 18

Answers Include:

Posttranslational modification (phosphorylation)

Dimerization

Subcellular localisation

Allosteric effectors

Intramolecular interaction

Multimer function

(Slide 18)

Question 19

What is an example of a pathway which has kinases which are regulated by phosphorylation?

Answer 19

MAPK pathway

(Slide 19)

Question 20

What are the steps of the MAPK pathway?

Answer 20

1. A growth factor like EGF binds to bind to their specific receptor tyrosine kinases (RTKs) on the cell surface.
2. Binding induces dimerization and auto-phosphorylation via tyrosine residues on their intracellular domains
3. Phosphorylated RTKs recruit adaptor proteins like Grb2 (Growth Factor Receptor-Bound Protein 2)
4. Grb2 binds SOS (Son of Sevenless), a guanine nucleotide exchange factor (GEF), which activates Ras by facilitating exchange of GDP for GTP
5. GTP-bound Ras activates Raf (MAPKKK), a serine/threonine kinase and the first kinase in the MAPK cascade
6. Raf phosphorylates and activates MEK (MAPKK), which phosphorylates ERK (MAPK).
7. Activated ERK translocates to the nucleus, and phosphorylates transcription factors, altering gene expression, or stay in the cytoplasm and phosphorylate other substrates, such as cytoskeleton proteins, phospholipases or other kinases

(Slide 20)

Question 21

What is the classical MAPK pathway responsible for regulating?

Answer 21

Cell proliferation and survival, as well as cell differentiation,

(Slide 20)

Question 22

What is the “D” / DEJL motif in MAP kinases and what does it do?

Answer 22

It is an accumulation of basic amino acids as the N or C terminal resulting in a hydrophobic motif composed of leucine (L), isoleucine (I) or valine (V) amino acids separated by another random amino acid residue (X), resulting in: (L/I/V) - X - (L/I/V).

It is located up to 20 amino acids away from the phosphorylation site and allows the kinase to bind proteins with a complementary L, I or V residue.

(Slide 21)

Question 23

What is the “DEF” motif on MAP kinases and what does it help do?

Answer 23

It is comprised of 2 phenylalanine residues (F) separated by a random amino acid residue (X) and often followed by a proline (P) resulting in: F-X-F-P.

It is located 6-10 amino acids away from the phosphorylation site (so near the DEJI motif).

It helps the kinase bind ERK (MAPK) or other MAP kinases, as well as scaffold proteins or substrates with a complimentary F-X-F sequence

(Slide 21)

Question 24

What is the motif for the phosphorylation (site P)?

Answer 24

It is a proline (P) followed by another random amino acid (X), followed by a serine or threonine (S or T) and then another proline resulting in: P-X-(S/T)-P

Note: This sequence starts 2 amino acids before the phosphorylation site and ends 1 residue after it, making the S/T residue in this sequence the phosphorylation site.

Note2: This is the site where the kinase is phosphorylated by another kinase, activating it, not where it phosphorylate substrates

(Slide 21)

Question 25

What is the docking groove?

Answer 25

A specific region on the protein, outside of the active catalytic site, that interacts with short, linear sequences (docking motifs) found in substrate proteins or regulatory proteins. It usually contains the D and DEJL motifs (Slide 22)

Question 26

How does dimerization of a MAP kinase occur and how does this affect localisation?

Answer 26

Phosphorylation occurs at threonine and tyrosine residues in the activation Phosphotyrosine interacts with the αC-helix. Phosphorylation at the threonine then results in a change in conformation which extends the C-terminal, exposing a hydrophobic surface The MAP kinase then forms a homodimer with another MAP kinase with this surface binding to the same surface on another kinase Homodimer formation via this surface results in localisation of the MAP kinase in the nucleus (Slide 23)

Question 27

Why is dimerization and localisation important for MAP kinase (and some other kinases)?

Answer 27

Dimerization ensures that the MAP kinases are properly activated and have catalytic activity. Localisation directs the MAP kinase to the right cellular component (the nucleus here) and ensures they regulate gene expression and other cellular processes correctly in response to signals. These also enhance specificity and efficiency. (Slide 23)

Question 28

How is the active site exposed in ERK (MAPK)?

Answer 28

Once the threonine residue is phosphorylated, the same conformational change which results in the extension of the C-terminal also exposes the active site of the kinase (Slide 24)

Question 29

How can phosphorylation be mimicked in some kinases and what does this result in?

Answer 29

By replacement of the corresponding amino acid (such as a threonine of tyrosine) with a negatively charged amino acid such as aspartate (D) or glutamate (E) resulting in a constitutively active kinase (Slide 24)

Question 30

What are GRK2 and p38 MAPK?

Answer 30

GRK2 stands for G-protein-coupled receptor kinase 2. p38 MAPK is a different type of MAPK (as opposed to ERK), which is responsible for regulating inflammatory mediators and stress responses. It also mediates cell cycle arrest and mediates production of cytokines such as TNF-α, IL-1, and IL-6. It is activated by a different MAPKK and MAPKKK than ERK is. (Slide 25)

Question 31

How does GRK2 interact with p38 MAPK?

Answer 31

It phosphorylates p38 at threonine-123 located at the edge of the "docking groove". The phosphorylated threonine-123 prevents binding and therefore prevents activation of p38 by MKK6, inactivating phosphorylation (Slide 25)

Question 32

How does PTPpep function in allosteric regulation of MAPK?

Answer 32

PTPpep (peptide of the hematopoietic tyrosine phosphatase), binds to MAPK and makes protected phosphorylation sites of the activation loop accessible to the solvent via a conformational change, allowing phosphorylation to occur (Slide 29)

Question 33

How does cyclin function in allosteric regulation in cyclin-dependent kinases (CDKs)?

Answer 33

It serves as an adaptor (a molecule or protein that facilitates or mediates the interaction between two other molecules) for substrate recognition by binding to the R-X-L-docking groove and changing the conformation of the CDK, activating it (Slide 30)

Question 34

What are EGR receptor tyrosine kinases?

Answer 34

They are transmembrane glycoproteins in which monomers are auto-inhibited and activated by ligand-induced dimerization (Slide 31)

Question 35

How does ligand-induced dimerization EGF receptor tyrosine kinases (EGFRs) occur and how does this activate the receptors?

Answer 35

A ligand binds to an EGFR, which causes a conformational change which exposes a dimerization interface on the receptor's extracellular domain. This promotes homodimerization of heterodimerization with a different receptor of the same family. Dimerization brings the intracellular tyrosine kinase domains closer to each other, meaning transphosphorylation can occur. This creates docking sites for downstream signalling proteins. (Slide 31)

Question 36

What is transphosphorylation?

Answer 36

Where one receptor's intracellular tyrosine kinase domain phosphorylates tyrosine residues on the other receptor's intracellular tyrosine domain (Slide 31)

Question 37

What do EGF receptor tyrosine kinases (EGFRs) regulate?

Answer 37

Cell growth, proliferation, differentiation, survival, and migration (Slide 31)

Question 38

What do c-Abl tyrosine kinases regulate?

Answer 38

Cell growth, survival, apoptosis, DNA repair, cytoskeleton dynamics, and cell migration (Slide 33)

Question 39

How does intramolecular interaction regulate activation of c-Abl tyrosine kinases?

Answer 39

In its inactive state, c-Abl is autoinhibited by interactions between its SH3, SH2 and kinase domains. Stress signals or other activation signals (such as growth factor receptors or cytokines), disrupt these intramolecular interactions, resulting in the release of the SH3-domain, the phosphorylation of tyrosines 412 (on the activation loop) and 245 (an SH2-kinase linker) and release of the myristoyl group, which all lead to a fully active c-Abl kinase. (Slide 33)

Question 40

What does Ca2+/calmodulin-dependent protein kinase II regulate?

Answer 40

Cardiac function and synaptic plasticity and memory as well as muscle contraction (Slide 35)

Question 41

How is Ca2+/calmodulin-dependent protein kinase II (CaMK II) activated and regulated by multimer function?

Answer 41

In its inactive state, the regulatory domain of each subunit of CaMK II interacts with the kinase domain of the same subunit, blocking its active site and prevents the kinase from phosphorylating any targets. Calmodulin binds Ca2+ and subsequently binds the regulatory domains CaMK II, disrupting the auto-inhibitory interaction of CaMK II and activating the kinase. Activation leads to trans-autophosphorylation between the monomer subunits (multimer function), which prevents the regulatory domain from binding to the kinase domain, resulting in the activity of the kinase becoming independent from Ca2+ and calmodulin. The autophosphorylation "mimics" the calcium signal even after calcium dissociates (Slide 35)

Question 42

What does phosphorylation at threonine-286 of Ca2+/calmodulin-dependent protein kinase II (CaMK II) do?

Answer 42

It greatly (13,000x) increases CAMK II's affinity for calmodulin, which keeps calmodulin tethered to CAMK II, enhancing its activation and maintaining it for longer. (Slide 35)

Question 43

What are 2 types of signal duration?

Answer 43

Transient activation and permanent activation (Slide 38)

Question 44

What is transient activation?

Answer 44

When a kinase is activated for a short amount of time. This is regulated by phosphorylation / dephosphorylation cycles. Once a kinase is activated by phosphorylation, the activation is often terminated quickly by phosphatases that remove the phosphate groups, returning the kinase to an inactive state. Negative feedback loops can also regulate this and sometimes receptor desensitisation or even internalisation can occur. (Slides 37 and 38)

Question 45

What is permanent activation?

Answer 45

The sustained activation of a kinase or signalling pathway, often resulting in long-term change in the cell. This type of activation may occur in response to chronic stimuli or persistent signalling. This can be regulated by auto-phosphorylation (such as in CaMK II), positive feedback loops, scaffold proteins, signal amplification, or by kinase degradation being delayed. (Slide 38)

Question 46

What are 3 examples of classes of diseases which are characterised by the deregulation of kinase activity?

Answer 46

Inflammatory diseases (such as rheumatoid arthritis) Various forms of cancers (such as chronic myeloic leukaemia or various tumours) Neurodegenerative diseases (such as Alzheimer's or amyotrophic lateral sclerosis) (Slide 39)

Question 47

What are 2 examples of compounds approved to inhibit kinases in order to treat cancers or other diseases?

Answer 47

Answers Include: Imatinib - chronic myeloic leukaemia (CML) Dasatinib - gastrointestinal storma tumours (GIST) and CML Gefitinib and Erlotinib - non-small cellular lung carcinoma Sorafenib and Sunitinib - kidney carcinoma (plus GIST for sunitinib) Fasudil - cerebral vasospasm Rapamycin - used for immunosuppression during kidney transplantation (Slide 40)