Transcript Cell Biology - Models NF

Cell Biology - Models
The NF-kB/IkB System
Yurochko
February 19-20, 2008
Lecture Goal & Outline
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Goal:
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To introduce you to a model of signal transduction
and specifically examine a signaling pathway. The
pathway being the NF-kB/IkB regulatory pathway.
Outline:
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NF-kB
IkBs
IKKs
Upstream Regulators
NF-kB Biological Implications:
Human Disease
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Diseases associated with a dysregulation of
NF-kB.
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Atherosclerosis
Asthma
Arthritis
Cancer
Diabetes
Inflammatory bowl disease
Stroke
Viral Infections (AIDS)
NF-kB Biological Implications:
Health
 NF-kB
regulation is essential to many
aspects of our health including:
 cellular
development
 cellular survival
 the immune system
What Are We Talking About??
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Quick Overview of
the NF-kB/IkB
Signaling Pathway
Simple (?) Model Of The Known
Players In NF-kB Induction
IL-1
Virus
TNF
PMA
IL-1R1
TNFR1
Other
IRAK
?
Ras
TRADD
Cell
Memb.
PKC
TRAF2
TRAF6
NIK
MEKK1
MAPK
IKK ( and )
pp90rsk
IkB Phosphorylation
NF-kB/IkB
IkB Degradation
Free NF-kB
NF-kB Translocation
NF-kB Responsive Genes
Nuclear
Memb.
The Players
 NF-kB
 The
IkBs
 The IKKs
 Other Upstream Regulators
History of NF-kB
 Discovered
in 1986 in the laboratory of
Dr. David Baltimore.
 Found as a nuclear factor in B cells.
 Found to transactivate the kappa light
chain promoter.
 Later found to activate many genes.
What is NF-kB
NF-kB is a heterodimeric transcription factor from
the rel-family of transcription factors.
 Classic NF-kB is made up of two subunits termed
p50 and p65.
 Other members include c-rel, RelB, p52, as well
as the two precursors p105 and p100.
 Multiple subunits all interact to form a variety of
factors with different apparent functions.
 Evolutionarily conserved family of proteins.
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The NF-kB and IkB Family
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Schematic of the
NF-kB/IkB families
 Details KEY
features
Qu ickTime™ and a
TIFF (Uncompressed) deco mpressor
are nee ded to see this picture.
DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.
NF-kB as a Transcription Factor
 Contains
a DNA binding domain and a
transactivation domain.
 The p65 subunit contains the transactivation
domain and the p50 subunit contains the
DNA binding domain.
 The NF-kB subunits contains a relhomology domain.
RHD - Defines this Family
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Common to all members of the NF-kB family.
 Is ~ 300 a.a. domain.
 Is a multifunctional domain.
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Controls NF-kB dimerization.
Allows interaction with the IkBs.
DNA Binding.
Contains the NLS.
Picture of Rel-Proteins
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View of NF-kB
binding DNA.
NF-kB
DNA
Transcriptional Regulation by
NF-kB -- Mechanisms
Binds to a unique sequence found in the kBresponsive promoters (5’-GGGRNNYYCC-3’).
 c-Rel, RelB, and RelA (p65) contain
transactivation domains.
 The NF-kB family members interact with other
transcription factors and members of the basal
transcriptional machinery.
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NF-kB interacts with HMG-I, bZIP proteins, Sp1,
C/EBP
c-Rel and RelA interact with TBP
RelA interacts with TFIIB
Specificity
- Transcription Factor
Are there other mechanisms of
specificity?????????
From Science, 2004, 306:632-635
Reminder: How a TF works
http://life.nthu.edu.tw/~lslpc/StrucBio/chapter9/chapter9_2.html
Reminder: How a TF works
Enhancer
NF-kB
IID
TATA BOX
-25
Diagram based on and adapted from Struhl, K., Cell 84: 179-182
Initiator
Genes Regulated by NF-kB
Good vs. Bad
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The Good
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Immune Responsive Genes
Cytokine Genes
Adhesion Molecules
Transcription Factors
Growth Factors and Proliferative Genes
The Bad
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Viral Promoters
Growth Factors and Proliferative Genes
Inflammatory Genes
Is NF-kB really Important????
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Through the use of Knock-Out animals the
critical role NF-kB plays in health has been
demonstrated.
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p65 KO -- embryonic lethality.
p50 KO -- develops normally, but has B cell
immune defects.
RelB KO -- develops normally, but has immune
defects and changes in hematopoiesis.
c-rel KO -- develops normally, but B cells and T
cells are unresponsive to certain activating
signals.
Mechanism: NF-kB Activation
NF-kB activity is regulated by a family of
inhibitors termed IkB which include IkB, IkB,
IkB, the p105 and p100 precursors, and Bcl-3.
 Specifically, the IkBs binds to NF-kB and keeps it
sequestered in an inactive state in the cytosol.
 Following cellular activation (by many different
stimuli (cytokines, mitogens, viral infection, etc.),
a complex signaling cascade is initiated which
ultimately frees NF-kB from IkB allowing it to
translocate to the nucleus and transactivate kBresponsive elements.
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The IkBs
 There
are two main IkBs
 IkB
 IkB
 There
are also other less studied IkBs
or IkB like molecules.
 IkB
 The
C-terminal portions of p100 and p105.
 BCL3
The NF-kB and IkB Family
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Schematic of the
NF-kB/IkB families
 Details KEY
features
Qu ickTime™ and a
TIFF (Uncompressed) deco mpressor
are nee ded to see this picture.
DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.
IkB vs. IkB
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IkB is the prototypic IkB.
 We first discovered it in 1990.
 It is a 37 kDa protein.
 Binds to NF-kB and blocks its NLS.
 Regulates the rapid release of NF-kB and its
rapid down regulation.
 Also contains a nuclear export signal which is
important in the removal of NF-kB from the
nucleus.
IkB vs. IkB
 IkB
is a 46 kDa protein.
 First discovered in 1995.
 Blocks the NLS of NF-kB.
 Regulates the persistent release of NFkB.
 Also appears to protect NF-kB from the
negative effects of IkB
 Specificty????
Mechanism: IkB Regulation
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The IkBs contain critical serine residues.
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IkB -- S32 & S36
IkB -- S19 & S23
These serines are the targets of upstream
serine kinases termed IKKs (IkB Kinases).
 Following phosphorylation, the IkBs are
ubiquitinated and targeted for degradation by
the 26S proteasome.
IkB
Degradation
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Schematic of the
regulatory serines and a
quick look at the
ubiquitination event
(occurs at lysines 21
and 22 on IkB).
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E1 - ubiquitin-activating
enzyme
E2 - ubiquitin-conjugating
enzyme
E3 - ubiquitin-ligating
enzyme
Is IkB really Important????
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IkB KO -- born normally but die of a wasting
disease by day 7.
The IKKs
 There
is an IKK complex composed of
three known subunits. May include
others, as the complex is 700-900 kDa.
 Two of the members, IKK and IKK
are catlytic subunits (85 & 87 kDa,
respectively).
 The third member, IKKg (NEMO), is a
regulatory subunit (48 kDa).
The IKKs
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IKK and IKK have a very similar primary
structure (52% a.a. identity, ~70% DNA
identity). Contain the same domains.
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a leucine zipper (for protein-protein interactions),
a helix-loop-helix domain (regulatory function),
a kinase domain (functional properties).
IKKgdoes not contain a catalytic domain and
is very different from IKK and IKK.
Probably interacts with IKK and IKK as a
dimer or a trimer.
Schematic of the IKKs
Häcker and Karin, 2006, Sci. STKE, 357:1-19.
www.stke.org/cgi/contents/full/2006/357/re13
IKK Mechanisms of Action
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A model of how IKK
activity is regulated
(both up- and downregulated).
 Controlled by
phosphorylation
(kinase dependent
event).
Häcker and Karin, 2006, Sci. STKE, 357:1-19.
www.stke.org/cgi/contents/full/2006/357/re13
Are the IKKs really Important?
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In Mice:
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IKK KO -- born alive but died shortly after birth. Showed
severe muscular and skeletal defects. Had normal activation
of NF-kB following proinflammatory stimuli.
IKK KO -- embryonic lethality (similar to the p65 KO
animal).
IKKg KO -- embryonic lethality (similar to the p65 & IKK KO
animal).
Suggests what???????????
Defects in IKK KOs
Hu et al., 1999, Science 284:316-320
More Defects in IKK KOs
Hu et al., 1999, Science 284:316-320
Defects in IKK KOs
Li et al., 1999, Science 284:321-325
Are the IKKs really Important?
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In humans, there is a diagnosed genetic
defect in which IKKg is absent. (Called
Incontinentia Pigmenti)
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In males - embryonic lethality (usually)
In females -- congenital disorder of teeth, hair, and
sweat glands, death usually occurs early in life.
Incontinentia Pigmenti
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Rare familial X-linked dominant condition (Xlinked recessive trait (chromosomal locus
Xq28)).
 Characteristics include
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Skin lesions
Hair, eye, teeth, and nail abnormalities
Osteosclerosis
Immune system disorders (immunodeficiency
resulting in recurrent infections)
Some males do survive for several years (usually
have a milder genetic abnormality)
Affected Males
IKKg/NEMO - Another Role
 IKKg
or NEMO can function as a bridge
to the interferon signaling pathway
IKKg also has the capacity to
regukate signal transduction pathways
independent of its role in the regulation
of NF-kB activation
 Thus
At this point, what do we know?
Other Upstream Regulators
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The are many upstream regulators described
in the literature. How each upstream kinase
fits in, is unclear, especially in regards to
specific signaling.
 IKK regulation appears to be a point of
convergence for a number of different
signaling pathways.
 Some of the upstream players include:
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NIK (NF-kB Inducing Kinase)
MEKK1 (A MAP3K)
Ras/Raf
Others
More Details
Possible Specificity????
 NIK
seems to preferentially activate
IKK.
 MEKK1 seems to preferentially activate
IKK.
 Suggests what????
What Activates NF-kB?????
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Cytokines
 Growth Factors
 Cell Adhesion
 Viral Infection
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Thus a Receptor-Ligand mediated event.
One Last Concluding Figure
Molecular Cell Biology; 4th Edition
Other mechanisms of specificity?????
Everything you ever learned in
one cartoon!
IMAGES FROM: G. Orphnides and D. Reinberg 2002, Cell 108: 439-451
NF-kB Biological Implications:
Health & Human Disease

NF-kB regulation is essential to many aspects of our
health including:




Diseases associated with a dysregulation of NF-kB.


cellular development
cellular survival
the immune system
Atherosclerosis, Asthma, Arthritis, Cancer, Diabetes,
Inflammatory bowl disease, Stroke, Viral Infections (AIDS)
Thus, together this is a critical pathway and one that
warrants much attention to understand its role in
human pathobiology.
NF-kB Biological Implications:
Health
 NF-kB
regulation is essential to many
aspects of our health including:
 cellular
development
 cellular survival
 the immune system