Lecture 12 - Lymphocyte Activation Flashcards
Transcription factors activated by TCR activation
1)
2)
3)
1) NFkB
2) NFAT
3) AP-1
Function of NFkB, NFAT and AP-1
Transcription factors
All required to bind to promotor for expression of certain genes, EG: IL-12
Components of complete TCR complex 1) 2) 3) 4) 5)
1) TCR
2) CD3 dimers
3) CD8/CD4
4) Lck
5) ZAP-70
Types of CD3 dimers
1)
2)
3)
1) Epsilon/Delta dimer
2) Gamma/Delta dimer
3) Zeta homodimer
ITAM
Immunoreceptor Tyrosine-based Activation Motif
ITAM structure
Yxx[L/V]x(6-9)Yxx[L/V]
Where x is any amino acid
What phosphorylates Y residues in ITAMS?
Src family kinases
EG: Lck, Fyn
Src family regulation
Activation - CD45 removes phosphate from kinase domain
Inactivation - C-terminal Src Kinase (Csk) adds phosphate to kinase domain
What phosphorylates Src kinases?
C-terminal Src kinase (Csk)
Phosphorylates C-terminal Y of Src
What removes a phosphate from Src kinase?
CD45
Examples of Src kinases
Lck
Fyn
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
Lck location
Cell membrane bound
Without CD4/CD8, not complexed with TCR complex
What brings Lck into contact with TCR/CD3 complex?
CD4/CD8 will bind both B2/A3 of TCR (extracellular) and Lck (intracellular)
Lck function
Phosphorylates ITAMs on CD3 complex
Src family strucutre
SH3 - SH2 - Kinase domain
Syk family structure
SH2 - SH2 - Kinase
What is ZAP-70?
A Syk family kinase
What recruits ZAP-70?
Phosphorylated ITAM of CD3zeta
What does the phosphorylated ITAM of CD3zeta recruit?
ZAP-70
ZAP-70 function
Phosphorylates LAT, phospholipase Cgamma
What activates Syk family kinases?
SH2 domain interaction with phosphotyrosines on other proteins
Example of a Syk family kinase
ZAP-70
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
LAT
Linker of T cell Activation
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
Two signalling pathways initiated by phosphorylation of LAT
1) Phospholipase C gamma (PLCgamma)
2) MAP kinase
What activates phospholipase C gamma and MAP kinase?
Phosphorylated LAT
What phosphorylates LAT?
ZAP-70 bound to TCR proximal signalling complex
What activates phospholipase C gamma?
ZAP-70 and Ita, when phospholipase C is bound to LAT
Phospholipase C gamma function
Catalyses the hydrolysis of membrane phospholipids
Phospholipase C gamma function in lymphocyte activation
Cleaves PIP2 into DAG and IP3
PIP2
Phosphatidyl inositol bisphosphate
Cleaved into DAG and IP3 by PLCgamma
DAG
Diacylglycerol
Product of PLCgamma cleaving PIP2
IP3
Inositol trisphosphate
Product of PLCgamma cleaving PIP2
Diacylglycerol function
DAG, Ca2+ activate protein kinase C
Inositol trisphosphate function
Increases intracellular Ca2+
This activates calcineurin
Protein kinase C function
NFkB activation
Calcineurin function
NFAT activation
What is NFkB?
A transcription factor
What is NFAT?
A transcription factor
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
IkB function
Binds to NFkB dimer in the cytoplasm
Prevents NFkB entry into the nucleus
What kind of regulation are NFkB and NFAT subject to?
Location-dependent regulation
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
Calcineurin
A phosphatase
Activated by elevated intracellular [Ca2+]
Cyclosporine function
Binds to calcineurin, prevents it from dephosphorylating NFAT
Immunosuppressant
Clinical use of cyclosporine
Transplants
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)
Example of a guanine nucleotide exchange factor
SOS
Example of a MAP kinase kinase kinase
Raf
Example of a MAP kinase kianse
Mak
Example of a MAP kinase
Erk
How does TCR binding activate MAP K?
1)
2)
3)
1) Phosphorylated LAT recruits Grb2
2) Grb2 recruits SOS
3) SOS activates ras GTPase
Which gene promotor does Elk bind to?
AP-1
Promotes AP-1 transcription
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
