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Cellular and Molecular Immunology > 12. Lymphocyte signalling 4: signal transduction > Flashcards

12. Lymphocyte signalling 4: signal transduction Flashcards

1
Q

What type of signalling is Ras?

A

MAP kinase cascade

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2
Q

What happens once Ras is activated?

A
  1. Ras is activated by a GEF like RasGRP.
  2. This allows Ras to cause a conformational change in Raf so it can autophosphorylate to activate.
  3. Raf is a MAPKKK
  4. Raf phosphorylates Mek which is a MAPKK.
  5. MEK phosphorylates Erk which is a MAPK.
  6. Erk generates the active transcription factor fos/jun.
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3
Q

What different serine threonine kinases are in MAP kinase pathways?

A
  1. MAP kinase
  2. MAP kinase kinase
  3. MAP kinase kinase kinase
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4
Q

What is a MAP kinase cascade?

A

A sequence of phosphorylation events of kinases that is triggered by a conformational change and cause a change in transcription.

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5
Q

What is the transcription factor that results from Ras signalling?

A

Jun and fos form that active dimer AP-1 that can bind promoters.

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6
Q

What are the 3 main MAP kinase pathways in every cell?

A
  1. Ras pathway ending with Erk
  2. Stress-activated MAPK pathway ending in a JNK kinase.
  3. TNF activated MAPK pathway ending in p38.
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7
Q

What is the common principle of all MAP kinase pathways?

A

A small GTPase activates a series of MAP kinases which causes a change in transcription.

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8
Q

What is the whole process from TCR signalling to Ras activation and transcriptional change?

A
  1. Lck phosphorylates ITAMs that recruits ZAP70
  2. ZAP70 phosphorylates the adaptor protein LAT
  3. LAT recruits SLP76 through phos tyrosines and SH2 domain binding
  4. SLP76 recruits ITK and PLCy
  5. ITK activates PLCy through phosphorylation
  6. PLCy hydrolyses PIP2 in DAG and IP3
  7. DAG activates GEFs Sos and RasGRP
  8. Ras is activated when bound to GTP as Ras changes conformation.
  9. This causes a change in conformation of Raf and allows it to autophosphorylate and activate.
  10. Raf phosphorylates Mek to activate it.
  11. Mek phosphorylates Erk to activate it
  12. Erk generates the active transcription factor AP-1.
  13. AP-1 is a dimer of Jun/fos
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9
Q

How are Mek inhibitors used in the clinic?

A
  1. Use to treat cancers with BRaf mutations.
  2. 4 different Mek inhibitors.
  3. Used in combinations with other treatments.
  4. Cancers can develop resistance to these inhibitors
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10
Q

What is NF-kB?

A

A widely used transcription factors in immune cells.

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11
Q

What does activation of NF-kB start with?

A

The cleavage of PIP2 into DAG and IP3 by PLC. DAG can activate NF-kB.

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12
Q

What is the protein kinase C family of proteins?

A
  1. It has lots of members that are critical for the activation of NF-kB.
  2. They are conserved through cells.
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13
Q

What Protein kinase C is important for NF-kB activation?

A
  1. PKC theta.
  2. PKC theta is a sub family of PKC.
  3. Has C1 domains that binds DAG for activation.
  4. Has a C2 like domain
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14
Q

What is important for NF-kB activation?

A

Co-stimulation from CD28

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15
Q

What TCR signalling is needed for NF-kB activation?

A
  1. TCR signalling causing Lck, ZAP70, LAT and SLP76 to recruit PLCy
  2. PLCy generates DAG.
  3. DAG binds to PKC theta and activates it.
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16
Q

What co-stimulatory signalling from CD28 is needed to activate NF-kB?

A
  1. CD28 engagement recruited PI 3-kinase through the YXXM ITAM.
  2. PI 3-kinase converts PIP2 to PIP3 at the plasma membrane.
  3. PIP3 recruits PDK1
  4. PDK1 phosphorylates PKC theta to fully activate it.
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17
Q

How does PIP3 recruit PDK1?

A

PDK1 contains a PH domain that can bind PIP3

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18
Q

What 2 signals are needed to activate PKC theta?

A
  1. DAG binding to the C1 domain triggered from TCR signalling.
  2. Phosphorylation from PDK1 triggered from CD28 signalling.
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19
Q

What does activated PKC theta do?

A

It activates the CARMA1/BCL10/MALT1 large signalling complex.

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20
Q

What is the function of the CARMA1/BCL10/MALT1 complex?

A

This complex activates a kinase complex called the inhibitor of NF-kB kinase complex (IKK).

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21
Q

What does IKK do?

A
  1. It phosphorylates the inhibitor of NF-kB
  2. This flags it for degradation.
  3. And releases NF-kB to be able to enter the nucleus.
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22
Q

What do mutations in the NF-kB cause?

A
  1. Severe immunodeficiency
  2. eg. Mutations in the gamma subunit of NF-kB of the IKK complex
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23
Q

How is ubiquitin used to flag proteins for degradation?

A
  1. Ubiquitin has a number of different lysine residues and ubiquitin chains are formed by binding to different residues.
  2. chains made by attaching to the K48 residue flag proteins for degradation.
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24
Q

What ubiquitin signalling is used in NF-kB activation?

A
  1. ubiquitin chains made by binding the K63 residues bind to TRAF6.
  2. This forms a scaffold for NF-kB activation
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25
Q

How does TNF receptor signalling contribute to NF-kB activation?

A
  1. TRAF6 is a signalling transducer that is associated with stimulatory TNR receptors.
  2. The stimulatory TNF receptor generates the K63 ubiquitin chains on the TRAF6
  3. These TRAF6 interact with the CARMA/BCL10/MALT1 complex.
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26
Q

What 3 signals are integrated and required for NF-kB activation?

A

It is a focal point of co-stimulatory signalling.
1. TCR signalling via DAG, PKC theta, CARMA1/Bcl10/MALT1
2. CD28 signalling via PI-3 kinase, PKC theta, CARMA1/Bcl10/MALT1
3. TNF signalling via TRAFs

27
Q

What is the structure of NF-kB?

A
  1. They are heterodimers.
  2. the most common is the p50 and p65 subunits.
28
Q

How does NF-kB enter the nucleus?

A
  1. IKK complex phosphorylates the inhibitor of NF-kB.
  2. This flags the inhibitor for degradation and recruit the K48 ubiquitin chain.
  3. When the inhibitor is degraded it releases NF-kB as an active transcription factor.
29
Q

What are the 3 post translational modifications that activate transcription factors?

A
  1. Dephosphorylation
  2. Phosphorylation
  3. Protein degradation of an inhibitor
30
Q

What does the IL-2 promoter tell us about signal integration in T cell activation?

A
  1. IL-2 is a key growth factor that drives T cell expansion.
  2. The IL-2 promoter contains lots of different binding element for transcription factors.
  3. This includes NFAT, AP-1 and NF-kB.
  4. You need activation of all the different signalling pathways to get effective T cell activation and proliferation.
31
Q

Why is metabolism a key signalling target in T cells?

A
  1. Cell proliferation requires active metabolism to provide energy and substrate for division.
  2. This includes ATP and other molecules needed to synthesise macromolecules.
  3. T cell proliferation is key for function and needs to expand their numbers massively in a short period of time.
  4. Cell proliferation is also central to cancer cells and this creates competition between Cancer and T cells.
32
Q

What are the key elements of metabolism?

A
  1. The generation of pyruvate from glucose.
  2. Glutamine being funnelled into the TCA cycle.
  3. Amino acid transport.
33
Q

Key elements in metabolism: conversion of glucose to pyruvate

A
  1. Glucose is converted to pyruvate then to lactate to make building block molecules needed for proliferation. This doesn’t produce a lot of energy
  2. Pyruvate can be entered straight into the TCA cycle to provide a lot of energy.
  3. The T cells need to choose which process to do.
  4. Most types of T cells choose generation of Lactate and small amounts of ATP.
34
Q

Key elements in metabolism: Glutamine entering the TCA cycle

A
  1. This is important for energy generation.
  2. Also makes important anabolic precursors like acetyl CoA to make lipids.
35
Q

How do T cells acquire biomolecules they cannot make?

A

Through uptake. This includes glucose and amino acids.

36
Q

What can altering metabolism do to T cells?

A

Alter the fate of differentiation of the T cell

37
Q

What are the key regulators in T cell metabolism?

A
  1. PIP3
  2. Akt
38
Q

How is the mTORC complex activated?

A
  1. PI 3-kinase is activated by signalling to generate PIP3.
  2. PIP3 recruits Akt
  3. Also required PDK1 to phosphorylate Akt.
  4. Akt phosphorylates key members of the mTORC complex.
39
Q

How is PIP3 generated for metabolic regulation?

A
  1. PI 3-kinase and PIP2 being converted to PIP3.
  2. Caused by Grb2 or CD28 signalling
40
Q

What is PIP3?

A
  1. A transmembrane protein
  2. Allows the recruitment of adaptor proteins like PDK1 or ITK
41
Q

What is Akt?

A
  1. A key regulator of metabolism
  2. A serine threonine kinase
  3. Present in every cell.
  4. Phosphorylates the mTORC complex
42
Q

What is mTORC?

A
  1. Mammalian target of rapamycin complex
  2. A key regulator of metabolism
43
Q

What is Rapamycin?

A
  1. A drug used to inhibit the immune system by inhibiting the mTORC complex.
  2. It suppresses key metabolic processes needed for immunity.
44
Q

What are the function of mTORC?

A
  1. Growth factor signalling to trigger cell proliferation.
  2. Detects the presence of the molecules needed for replication, like amino acids or nucleic acids. You cannot replicate if you don’t have enough material to make a new cell.
45
Q

What does mTORC use signalling to decide?

A
  1. Do I want to proliferate?
  2. Is everything available for the cell to proliferate?
46
Q

What are the 2 key regulators of mTORC?

A
  1. Amino acids.
  2. AMPK
  3. This tells mTORC if the cell has everything to proliferate.
47
Q

What is AMPK?

A
  1. AMP kinase
  2. It is activated by AMP
  3. AMP builds up if the cell cannot generate ATP. This means the cell has a lack of oxygen and ATP so is not ready to divide.
  4. This activates AMPK and inhibits mTORC.
48
Q

Why is the integration of different signals important?

A

For T cells need to respond to infection they need to be able to regulate gene expression and metabolism through multiple signalling mechanisms.

49
Q

What does mTORC complex regulate?

A
  1. Lipid synthesis
  2. Mitochondrial metabolism
  3. Glycolytic metabolism
  4. Protein synthesis
  5. Autophagy
50
Q

What is autophagy?

A
  1. A starvation mechanism that degrades some of the cytoplasmic contents to generate energy.
  2. It is turned off by mTORC.
51
Q

Where are mTORC complexes localised?

A
  1. By lysosomes
  2. This is where all amino acids and other molecules are taken into the cell.
  3. This is the best location to detect their presence.
52
Q

How are Akt inhibitors used in clinic?

A
  1. 1st Akt inhibitor called Capivasertip
  2. Treat HR+ and HER2- breast cancer
    with Akt mutations.
53
Q

What is the structure of cytokine receptors?

A
  1. They have a conserved signal transduction chain.
  2. Either beta chain, gp130, gamma chain.
  3. These associate with more specific receptor chains for each cytokine.
54
Q

What does mutation in the gamma chain cause?

A
  1. SCID - Severe Combined Immunodeficiency.
  2. The gamma chain forms part of the IL-2 receptor.
  3. IL2 is critical for T cell proliferation, so without the IL2R, there is no proliferation of T cells.
55
Q

What is the signalling mechanism of cytokine receptors?

A
  1. They cytokine receptor is engaged by the cytokine.
  2. JAK kinases are localised to the receptor.
  3. These JAK autophosphorylate on tyrosine residues to activate themselves.
  4. The JAK kinases phosphorylate STAT.
  5. STAT dimerises and can then enter the nucleus.
56
Q

What is STAT?

A
  1. A transcription factor.
  2. When phosphorylated they can dimerise and enter the nucleus.
57
Q

Why is the cytokine signalling mechanism so simple?

A
  1. There are lots of cytokines around
  2. They are abundant and unambiguous.
  3. It is simple as it doesn’t need to be sensitive.
58
Q

What do the different JAK and STAT do?

A

Each different STAT and JAK associate with different cytokine receptor eg JAK3 associates with IL-2

59
Q

What are the main signal transduction principles?

A
  1. Many signalling interactions are mediated by a small number of large domain families like SH2 domains.
  2. Adaptors allow the formation of large signalling complexes like LAT and LSP-76.
  3. Signalling is mediated by complexes with multiple components are mutually connected to each other like mTORC.
  4. Transcription factors need to be activated by post-translational modification like STAT phosphorylation.
  5. Small GTPases are universal signalling switches
60
Q

Which signalling pathways are not T cell specific?

A
  1. Calcium signalling through NFAT.
  2. MAP kinase signalling through jun/fos
  3. NF-kB activation through TRAF and IKK and signalling
61
Q

What are the main functional groups in signalling?

A
  1. Receptors with their intermediates like TCR, CD4, Lck, ZAP70, LAT and SLP-76.
  2. Co-stimulatory molecules like CD28, TNF receptors or cytokine receptor.
  3. Signal transduction through SLP76, Grb2, ITK and PLC.
62
Q

What is the key linker molecule in signalling?

A
  1. PIP2
  2. Its hydrolysis into DAG and IP3
  3. IP3 activating calcium signalling.
  4. DAG activating MAP kinase and PKC theta in NF-kB activation
63
Q

What are the key complexes in TCR signalling?

A
  1. CARMA/BCL10/MALT1 which co-ordinates co-stimulation signalling.
  2. mTORC for metabolic regulation
64
Q

What is the full process from TCR signalling through to transcriptional change with Nf-kB?

A
  1. Lck phosphorylates ITAMs that recruits ZAP70.
  2. ZAP70 phosphorylates the adaptor protein LAT.
  3. LAT recruits SLP76 through phos tyrosines and SH2 domain binding.
  4. SLP76 recruits ITK and PLCy
  5. ITK activates PLCy through phosphorylation.
  6. PLCy hydrolyses PIP2 in DAG and IP3.
  7. DAG binds PKC theta to activate it
  8. CD28 co-stimulation generates PIP3 using PI-3 kinase.
  9. PIP3 recruits PDK1 via its PH domain.
  10. PDK1 phosphorylates PKC theta to fully activate it.
  11. PKC theta phosphorylates key members of the CARMA1/BCL10/MALT1 complex to activate it.
  12. The CARMA1/BCL10/MALT1 complex activates the inhibitor of NF-kB kinase complex (IKK).
  13. IKK phosphorylates the inhibitor of NF-kB to flag it for degradation.
  14. This releases NF-kB.
  15. NF-kB can then enter the nucleus and change gene expression.
  16. TRAF 6 is also associated with the activation of IKK so co-stimulation with TNF is important for NF-kB activation.

Cellular and Molecular Immunology (17 decks)

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