Lecture 12 - Lymphocyte Activation Flashcards Preview

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Flashcards in Lecture 12 - Lymphocyte Activation Deck (56):
1

Transcription factors activated by TCR activation
1)
2)
3)

1) NFkB
2) NFAT
3) AP-1

2

Function of NFkB, NFAT and AP-1

Transcription factors
All required to bind to promotor for expression of certain genes, EG: IL-12

3

Components of complete TCR complex
1)
2)
3)
4)
5)

1) TCR
2) CD3 dimers
3) CD8/CD4
4) Lck
5) ZAP-70

4

Types of CD3 dimers
1)
2)
3)

1) Epsilon/Delta dimer
2) Gamma/Delta dimer
3) Zeta homodimer

5

ITAM

Immunoreceptor Tyrosine-based Activation Motif

6

ITAM structure

Yxx[L/V]x(6-9)Yxx[L/V]
Where x is any amino acid

7

What phosphorylates Y residues in ITAMS?

Src family kinases
EG: Lck, Fyn

8

Src family regulation

Activation - CD45 removes phosphate from kinase domain
Inactivation - C-terminal Src Kinase (Csk) adds phosphate to kinase domain

9

What phosphorylates Src kinases?

C-terminal Src kinase (Csk)
Phosphorylates C-terminal Y of Src

10

What removes a phosphate from Src kinase?

CD45

11

Examples of Src kinases

Lck
Fyn

12

How is Src kinase inhibited?
1)
2)
3)

1) C-terminal Src kinase phosphorylates kinase domain of Src kinase
2) Phosphorylated kinase domain bends around, binds to SH2 domain
3) SH2 domain is now prevented from binding to phosphotyrosines

13

Lck location

Cell membrane bound
Without CD4/CD8, not complexed with TCR complex

14

What brings Lck into contact with TCR/CD3 complex?

CD4/CD8 will bind both B2/A3 of TCR (extracellular) and Lck (intracellular)

15

Lck function

Phosphorylates ITAMs on CD3 complex

16

Src family strucutre

SH3 - SH2 - Kinase domain

17

Syk family structure

SH2 - SH2 - Kinase

18

What is ZAP-70?

A Syk family kinase

19

What recruits ZAP-70?

Phosphorylated ITAM of CD3zeta

20

What does the phosphorylated ITAM of CD3zeta recruit?

ZAP-70

21

ZAP-70 function

Phosphorylates LAT, phospholipase Cgamma

22

What activates Syk family kinases?

SH2 domain interaction with phosphotyrosines on other proteins

23

Example of a Syk family kinase

ZAP-70

24

Proximal signalling events
1)
2)
3)
4)
5)

1) MHC molecule provides a docking site for TCR/CD3 complex and CD4/CD8
2) CD4/CD8 binds Lck, bringing Lck into contact with CD3 complex
3) Lck phosphorylates ITAM on CD3zeta
4) ZAP-70 binds to phosphorylated ITAM of CD3zeta
5) Lck phosphorylates ZAP-70

25

LAT

Linker of T cell Activation

26

Function of LAT

Membrane bound protein which has many tyrosines that can be phosphorylated

Phosphorylated tyrosines provide docking sites for proteins with two SH2 domains

27

Two signalling pathways initiated by phosphorylation of LAT

1) Phospholipase C gamma (PLCgamma)
2) MAP kinase

28

What activates phospholipase C gamma and MAP kinase?

Phosphorylated LAT

29

What phosphorylates LAT?

ZAP-70 bound to TCR proximal signalling complex

30

What activates phospholipase C gamma?

ZAP-70 and Ita, when phospholipase C is bound to LAT

31

Phospholipase C gamma function

Catalyses the hydrolysis of membrane phospholipids

32

Phospholipase C gamma function in lymphocyte activation

Cleaves PIP2 into DAG and IP3

33

PIP2

Phosphatidyl inositol bisphosphate
Cleaved into DAG and IP3 by PLCgamma

34

DAG

Diacylglycerol
Product of PLCgamma cleaving PIP2

35

IP3

Inositol trisphosphate
Product of PLCgamma cleaving PIP2

36

Diacylglycerol function

DAG, Ca2+ activate protein kinase C

37

Inositol trisphosphate function

Increases intracellular Ca2+
This activates calcineurin

38

Protein kinase C function

NFkB activation

39

Calcineurin function

NFAT activation

40

What is NFkB?

A transcription factor

41

What is NFAT?

A transcription factor

42

NFkB regulation
1)
2)
3)

1) Normally, NFkB is a dimer in the cytosol, bound to Inactive kB (IkB)
2) Protein kinase C phosphorylates IkB, causing IkB to be degraded in proteosome
3) This allows NFkB to enter the nucleus, act as a transcription factor

43

IkB function

Binds to NFkB dimer in the cytoplasm
Prevents NFkB entry into the nucleus

44

What kind of regulation are NFkB and NFAT subject to?

Location-dependent regulation

45

NFAT regulation
1)
2)
3)
4)

1) NFAT is normally phosphorylated, which prevents it from entering the nucleus
2) When IP3 raises intracellular [Ca2+], calmodulin on calcineurin is activated
3) Calcineurin removes phosphate from NFAT
4) NFAT enters the nucleus, binds to promotor

46

Calcineurin

A phosphatase
Activated by elevated intracellular [Ca2+]

47

Cyclosporine function

Binds to calcineurin, prevents it from dephosphorylating NFAT

Immunosuppressant

48

Clinical use of cyclosporine

Transplants

49

Generic MAP kinase pathway
1)
2)
3)
4)
5)

1) Guanine nucleotide exchange factor (SOS) activates a small G protein (ras, rac)
2) Activated small G protein activates a MAP kinase kinase kinase
3) Map kinase kinase kinase activates MAP kinase kinase
4) MAP kinase kinase activates MAP kinase
5) MAP kinase activates transcription factors (EG: Elk)

50

Example of a guanine nucleotide exchange factor

SOS

51

Example of a MAP kinase kinase kinase

Raf

52

Example of a MAP kinase kianse

Mak

53

Example of a MAP kinase

Erk

54

How does TCR binding activate MAP K?
1)
2)
3)

1) Phosphorylated LAT recruits Grb2
2) Grb2 recruits SOS
3) SOS activates ras GTPase

55

Which gene promotor does Elk bind to?

AP-1
Promotes AP-1 transcription

56

Proportion of required AP-1 transcription caused by Elk

1/2
Other half is caused by costimulation by CD80, CD86, CD28
A checkpoint mechanism to prevent T cell overactivity