L11 - Lymphocyte Activation

Question 1

What are the activated transcription factors that can arise from TCR activation?

Answer 1

NFκB, NFAT and AP-1

Question 2

Tyrosine residues in CD3 ITAMs are phosphorylated by [—]-family kinase tyrosine kinases. What are some examples?

Answer 2

Src-family tyrosine kinases. E.g. Lck, Fyn

Question 3

Aside from the BCR what makes up the BCR signalling complex?

Answer 3

Igα and Igβ with ITAMs attached

Question 4

What are Src-family kinases regulated by?

Answer 4

Csk (C-terminal src kinase) and CD45

Question 5

What is / the role of Csk?

Answer 5

C-terminal src kinase

- Phosphorylates C-terminal tyrosine and inhibits Src kinase activation

Question 6

What is / the role of CD45?

Answer 6

Phosphatase that removes phosphate from Tyr505. Yin/yang of Csk
- Required for activation of Src kinases

Question 7

What is the structure of Lck?

Answer 7

SH3 domain, SH2 domain and Kinase domain with 2 tyrosine residues

Question 8

What are the tyrosine residues of Lck?

Answer 8

Tyr394 closer to SH2 domain and Tyr505 further down

Question 9

What is the terminal Tyrosine in Lck, what phosphorylates it and what does it do?

Answer 9

Tyr505 by Csk, blocks formation of active Src-kinase. Tyr505 binds to SH2 domain

Question 10

What phosphorylates Tyr394 and what is the result of this?

Answer 10

Tyr394 can autophosphorylate. Result of this is kinase domain is accessible / active. SH2 domain available to interact the phosphotyrosines on other proteins

Question 11

What happens if there is a mutation in Tyr505?

Answer 11

The molecule will be constitutively open, kinase site constitutively accessible, cannot be phosphorylayted

Question 12

What happens if there is a mutation in Tyr394?

Answer 12

The molecule becomes inactive

Question 13

What happens if there is a mutation in Tyr505 and mutation in kinase to render it ‘dead’

Answer 13

Clusters still appear, Lck is present in microclusters. Lck conformation rather than enzyme activity regulates cluster size

Question 14

What is the takeaway from the microclustering experiment?

Answer 14

Lck present in microclusters is dependent on Tyr394 being phosphorylated

Question 15

What kinase is associated with co-receptors CD8 and CD4?

Answer 15

Lck

Question 16

What happens if lck is in the presence of ITAMs?

Answer 16

Lck can phosphorylate ITAMs

Question 17

What family of kinase is ZAP-70?

Answer 17

Syk family kinase

Question 18

Where is ZAP-70 recruited to and what is it activated by?

Answer 18

Recruited to ITAM of CD3ζ

- Activated by lck

Question 19

What is ZAP-70’s role?

Answer 19

Phosphorylates LAT(substrate of ZAP-70) and phospholipase Cγ

Question 20

What domains does Syk family kinases have?

Answer 20

Two SH2 domains and a kinase domain

- Inactive until SH2 domains interact with phosphotyrosines

Question 21

What follows ZAP-70 recruitment onto CD3ζ?

Answer 21

Phosphorylated ITAMs of CD3ζ by lck. lck then phosphorylates ZAP-70

Question 22

What is LAT?

Answer 22

Linker of T cell activation

• Contains tyrosine residues that can be phosphorylated
• Phosphorylated tyrosines serve as docking site for other proteins/adaptors that contain SH2 domains

Question 23

What pathways can phosphorylation of LAT lead to?

Answer 23

• Activation of phospholipase Cγ

- Activation of the MAP kinase pathway

Question 24

What is LAT’s relation to PLCγ?

Answer 24

LAT serves as a docking site for PLCγ (binds to phosphotyrosines in LAT)

Question 25

What 2 molecules can activate PLCγ?

Answer 25

ZAP-80 itself or Itk

Question 26

What happens following PLCγ activation?

Answer 26

Catalyses hydrolysis of membrane phospholipids (especially PIP2)
- Activates 2 distinct pathways leading to DAG or IP3

Question 27

What final activation does production of DAG lead to?

Answer 27

Activation of NFκB

Question 28

What final activation does production of IP3 lead to?

Answer 28

Activation of NFAT

Question 29

DAG and Ca2+ activate what?

Answer 29

Protein Kinase C

Question 30

Where is NFκB found and what keeps it in its inactive state?

Answer 30

Cytoplasm and bound to IκB

Question 31

What does PKC do?

Answer 31

Activation of IκB kinase

Question 32

What does IκB Kinase do?

Answer 32

Phosphorylates IκB leading to proteolysis of IκB (by proteosome) leading to active NFκB to translocate to the nucleus leading to gene transcription

Question 33

What does IP3 do?

Answer 33

Increase intracellular Ca2+ concentration binding to calmodulin activating calcineurin

Question 34

What does calcineurin do?

Answer 34

Activates NFAT through phosphatase activity. Removes phosphate from NFAT activating it and allowing it to translocate to the nucleus

Question 35

What is the difference between inactive NFAT and inactive NFκB?

Answer 35

NFAT is phosphorylated in its inactive state

Question 36

What is the relevance of the NFAT pathway in relation to transplantation and immunosuppression?

Answer 36

Cyclosporine/FK-506 is an immunosuppressive agent. Binds to calcineurin blocking the phosphatase activity and hence NFAT translocation/activation

Question 37

How is the generic MAP kinase pathway initiated?

Answer 37

Small G proteins activated by guanine nucleotide exhcnage factors

Question 38

What does activated G protein activate and so on?

Answer 38

• Activated G protein activates MAP kinase kinase kinase
• MAP kinase kinase kinase activates MAP kinase kinase
• MAP kinase kinase activates MAP kinase
• MAP kinase phosphorylates transription factors which translocate to nucleus and activate gene transcription

Question 39

What are the other names for the following:
- G-protein
- MAP kinase kinase kinase
- MAP kinase kinase
MAP kinase
- Activated transcription factor

Answer 39

G-protein: Ras
MAP Kinase kinase kinase: Raf
MAP kinase kinase: Mek
MAP kinase: Erk
Activated transcription factor: Elk -> AP-1