Lecture 12 - Lymphocyte Activation

Question 1

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

Answer 1

1) NFkB
2) NFAT
3) AP-1

Question 2

Function of NFkB, NFAT and AP-1

Answer 2

Transcription factors

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

Question 3

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

Answer 3

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

Question 4

Types of CD3 dimers
1)
2)
3)

Answer 4

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

Question 5

ITAM

Answer 5

Immunoreceptor Tyrosine-based Activation Motif

Question 6

ITAM structure

Answer 6

Yxx[L/V]x(6-9)Yxx[L/V]

Where x is any amino acid

Question 7

What phosphorylates Y residues in ITAMS?

Answer 7

Src family kinases

EG: Lck, Fyn

Question 8

Src family regulation

Answer 8

Activation - CD45 removes phosphate from kinase domain

Inactivation - C-terminal Src Kinase (Csk) adds phosphate to kinase domain

Question 9

What phosphorylates Src kinases?

Answer 9

C-terminal Src kinase (Csk)

Phosphorylates C-terminal Y of Src

Question 10

What removes a phosphate from Src kinase?

Answer 10

CD45

Question 11

Examples of Src kinases

Answer 11

Lck

Fyn

Question 12

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

Answer 12

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

Question 13

Lck location

Answer 13

Cell membrane bound

Without CD4/CD8, not complexed with TCR complex

Question 14

What brings Lck into contact with TCR/CD3 complex?

Answer 14

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

Question 15

Lck function

Answer 15

Phosphorylates ITAMs on CD3 complex

Question 16

Src family strucutre

Answer 16

SH3 - SH2 - Kinase domain

Question 17

Syk family structure

Answer 17

SH2 - SH2 - Kinase

Question 18

What is ZAP-70?

Answer 18

A Syk family kinase

Question 19

What recruits ZAP-70?

Answer 19

Phosphorylated ITAM of CD3zeta

Question 20

What does the phosphorylated ITAM of CD3zeta recruit?

Answer 20

ZAP-70

Question 21

ZAP-70 function

Answer 21

Phosphorylates LAT, phospholipase Cgamma

Question 22

What activates Syk family kinases?

Answer 22

SH2 domain interaction with phosphotyrosines on other proteins

Question 23

Example of a Syk family kinase

Answer 23

ZAP-70

Question 24

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

Answer 24

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

Question 25

LAT

Answer 25

Linker of T cell Activation

Question 26

Function of LAT

Answer 26

Membrane bound protein which has many tyrosines that can be phosphorylated Phosphorylated tyrosines provide docking sites for proteins with two SH2 domains

Question 27

Two signalling pathways initiated by phosphorylation of LAT

Answer 27

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

Question 28

What activates phospholipase C gamma and MAP kinase?

Answer 28

Phosphorylated LAT

Question 29

What phosphorylates LAT?

Answer 29

ZAP-70 bound to TCR proximal signalling complex

Question 30

What activates phospholipase C gamma?

Answer 30

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

Question 31

Phospholipase C gamma function

Answer 31

Catalyses the hydrolysis of membrane phospholipids

Question 32

Phospholipase C gamma function in lymphocyte activation

Answer 32

Cleaves PIP2 into DAG and IP3

Question 33

PIP2

Answer 33

Phosphatidyl inositol bisphosphate | Cleaved into DAG and IP3 by PLCgamma

Question 34

DAG

Answer 34

Diacylglycerol | Product of PLCgamma cleaving PIP2

Question 35

IP3

Answer 35

Inositol trisphosphate | Product of PLCgamma cleaving PIP2

Question 36

Diacylglycerol function

Answer 36

DAG, Ca2+ activate protein kinase C

Question 37

Inositol trisphosphate function

Answer 37

Increases intracellular Ca2+ | This activates calcineurin

Question 38

Protein kinase C function

Answer 38

NFkB activation

Question 39

Calcineurin function

Answer 39

NFAT activation

Question 40

What is NFkB?

Answer 40

A transcription factor

Question 41

What is NFAT?

Answer 41

A transcription factor

Question 42

NFkB regulation 1) 2) 3)

Answer 42

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

Question 43

IkB function

Answer 43

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

Question 44

What kind of regulation are NFkB and NFAT subject to?

Answer 44

Location-dependent regulation

Question 45

``` NFAT regulation 1) 2) 3) 4) ```

Answer 45

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

Question 46

Calcineurin

Answer 46

A phosphatase | Activated by elevated intracellular [Ca2+]

Question 47

Cyclosporine function

Answer 47

Binds to calcineurin, prevents it from dephosphorylating NFAT Immunosuppressant

Question 48

Clinical use of cyclosporine

Answer 48

Transplants

Question 49

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

Answer 49

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)

Question 50

Example of a guanine nucleotide exchange factor

Answer 50

SOS

Question 51

Example of a MAP kinase kinase kinase

Answer 51

Raf

Question 52

Example of a MAP kinase kianse

Answer 52

Mak

Question 53

Example of a MAP kinase

Answer 53

Erk

Question 54

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

Answer 54

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

Question 55

Which gene promotor does Elk bind to?

Answer 55

AP-1 | Promotes AP-1 transcription

Question 56

Proportion of required AP-1 transcription caused by Elk

Answer 56

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