cell biology - models the nf- b/i b system yurochko february 19-20, 2008
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Lecture Goal & Outline Goal:
To introduce you to a model of signal transduction and specifically examine a signaling pathway. The pathway being the NF-B/IB regulatory pathway.
Outline: NF-B IBs IKKs Upstream Regulators
NF-B Biological Implications:Human Disease Diseases associated with a dysregulation of
NF-B. Atherosclerosis Asthma Arthritis Cancer Diabetes Inflammatory bowl disease Stroke Viral Infections (AIDS)
NF-B Biological Implications:HealthNF-B regulation is essential to many
aspects of our health including: cellular development cellular survival the immune system
What Are We Talking About?? Quick Overview of
the NF-B/IB Signaling Pathway
Simple (?) Model Of The Known Players In NF-B Induction
Free NF-B
NF-B Responsive Genes
NF-B/IB
IKK (α and β)
NIK
MEKK1
TRAF2TRAF6
TRADDIRAK
TNFR1IL-1R1
NF-B Translocation
IB Degradation
IB Phosphorylation
IL -1 TNF
Other
PKC
MAPK
pp90rsk
PMA
Virus
NuclearMemb.
CellMemb.
Ras?
History of NF-BDiscovered 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-B NF-B is a heterodimeric transcription factor from
the rel-family of transcription factors. Classic NF-B 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.
The NF-B and IB Family Schematic of the
NF-B/IB families Details KEY
featuresQuickTime™ and a
TIFF (Uncompressed) decompressorare needed to see this picture.
DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.
NF-B 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-B subunits contains a rel-homology domain.
RHD - Defines this Family Common to all members of the NF-B family. Is ~ 300 a.a. domain. Is a multifunctional domain.
Controls NF-B dimerization. Allows interaction with the IkBs. DNA Binding. Contains the NLS.
Transcriptional Regulation by NF-B -- Mechanisms Binds to a unique sequence found in the B-
responsive promoters (5’-GGGRNNYYCC-3’). c-Rel, RelB, and RelA (p65) contain transactivation
domains. The NF-B family members interact with other
transcription factors and members of the basal transcriptional machinery. NF-B 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
Diagram based on and adapted from Struhl, K., Cell 84: 179-182
Enhancer
InitiatorTATA BOX-25
IID
NF-B
Genes Regulated by NF-BGood vs. Bad The Good
Immune Responsive Genes Cytokine Genes Adhesion Molecules Transcription Factors Growth Factors and Proliferative Genes
The Bad Viral Promoters Growth Factors and Proliferative Genes Inflammatory Genes
Is NF-B really Important???? Through the use of Knock-Out animals the
critical role NF-B plays in health has been demonstrated. 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-B Activation NF-B activity is regulated by a family of inhibitors
termed IB which include IBα, IBβ, IB, the p105 and p100 precursors, and Bcl-3.
Specifically, the IBs binds to NF-B 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-B from IB allowing it to translocate to the nucleus and transactivate B-responsive elements.
The IBsThere are two main IBs
IBα IBβ
There are also other less studied IBs or IB like molecules. IB The C-terminal portions of p100 and p105. BCL3
The NF-B and IB Family Schematic of the
NF-B/IB families Details KEY
featuresQuickTime™ and a
TIFF (Uncompressed) decompressorare needed to see this picture.
DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.
IBα vs. IBβ IBα is the prototypic IB. We first discovered it in 1990. It is a 37 kDa protein. Binds to NF-B and blocks its NLS. Regulates the rapid release of NF-B and its
rapid down regulation. Also contains a nuclear export signal which is
important in the removal of NF-B from the nucleus.
IBα vs. IBβ IBβ is a 46 kDa protein.First discovered in 1995.Blocks the NLS of NF-B.Regulates the persistent release of NF-B.
Also appears to protect NF-B from the negative effects of IBα
Specificty????
Mechanism: IB Regulation The IBs contain critical serine residues.
IBα -- S32 & S36 IBβ -- S19 & S23
These serines are the targets of upstream serine kinases termed IKKs (IB Kinases).
Following phosphorylation, the IBs are ubiquitinated and targeted for degradation by the 26S proteasome.
IB Degradation
Schematic of the regulatory serines and a quick look at the ubiquitination event (occurs at lysines 21 and 22 on IBα). E1 - ubiquitin-activating
enzyme E2 - ubiquitin-conjugating
enzyme E3 - ubiquitin-ligating
enzyme
The IKKsThere is an IKK complex composed of
three known subunits. May include others, as the complex is 700-900 kDa.
Two of the members, IKKα and IKKβ are catlytic subunits (85 & 87 kDa, respectively).
The third member, IKK (NEMO), is a regulatory subunit (48 kDa).
The IKKs IKKα and IKKβ have a very similar primary
structure (52% a.a. identity, ~70% DNA identity). Contain the same domains. a leucine zipper (for protein-protein interactions), a helix-loop-helix domain (regulatory function), a kinase domain (functional properties).
IKK does not contain a catalytic domain and is very different from IKKα and IKKβ. Probably interacts with IKKα and IKKβ 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
A model of how IKK activity is regulated (both up- and down-regulated).
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? In Mice:
IKKα KO -- born alive but died shortly after birth. Showed severe muscular and skeletal defects. Had normal activation of NF-B following proinflammatory stimuli.
IKKβ KO -- embryonic lethality (similar to the p65 KO animal).
IKK KO -- embryonic lethality (similar to the p65 & IKKβ KO animal).
Suggests what???????????
Are the IKKs really Important? In humans, there is a diagnosed genetic
defect in which IKK is absent. (Called Incontinentia Pigmenti) In males - embryonic lethality (usually) In females -- congenital disorder of teeth, hair, and
sweat glands, death usually occurs early in life.
Incontinentia Pigmenti Rare familial X-linked dominant condition (X-
linked recessive trait (chromosomal locus Xq28)).
Characteristics include 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)
IKK/NEMO - Another Role IKK or NEMO can function as a bridge
to the interferon signaling pathway
Thus IKK also has the capacity to regukate signal transduction pathways independent of its role in the regulation of NF-B activation
Other Upstream Regulators 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: NIK (NF-B Inducing Kinase) MEKK1 (A MAP3K) Ras/Raf Others
More Details Possible Specificity????NIK seems to preferentially activate
IKKα.MEKK1 seems to preferentially activate
IKKβ.Suggests what????
What Activates NF-B????? Cytokines Growth Factors Cell Adhesion Viral Infection
Thus a Receptor-Ligand mediated event.
Everything you ever learned in one cartoon!
IMAGES FROM: G. Orphnides and D. Reinberg 2002, Cell 108: 439-451
NF-B Biological Implications:Health & Human Disease NF-B regulation is essential to many aspects of our
health including: cellular development cellular survival the immune system
Diseases associated with a dysregulation of NF-B. 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.
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