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Cell Biology - Models The NF- k B/I k B System. Yurochko February 19-20, 2008. Lecture Goal & Outline. Goal: To introduce you to a model of signal transduction and specifically examine a signaling pathway. The pathway being the NF- k B/I k B regulatory pathway. Outline: NF- k B I k Bs

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cell biology models the nf k b i k b system

Cell Biology - ModelsThe NF-kB/IkB System

Yurochko

February 19-20, 2008

lecture goal outline
Lecture Goal & Outline
  • Goal:
    • 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:
    • NF-kB
    • IkBs
    • IKKs
    • Upstream Regulators
nf k b biological implications human disease
NF-kB Biological Implications:Human Disease
  • Diseases associated with a dysregulation of NF-kB.
    • Atherosclerosis
    • Asthma
    • Arthritis
    • Cancer
    • Diabetes
    • Inflammatory bowl disease
    • Stroke
    • Viral Infections (AIDS)
nf k b biological implications health
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
What Are We Talking About??
  • Quick Overview of the NF-kB/IkB Signaling Pathway
the players
The Players
  • NF-kB
  • The IkBs
  • The IKKs
  • Other Upstream Regulators
history of nf k b
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 k b
What is NF-kB
  • 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 k b and i k b family
The NF-kB and IkB Family
  • Schematic of the NF-kB/IkB families
  • Details KEY features

DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.

nf k b as a transcription factor
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 rel-homology domain.
rhd defines this family
RHD - Defines this Family
  • Common to all members of the NF-kB family.
  • Is ~ 300 a.a. domain.
  • Is a multifunctional domain.
    • Controls NF-kB dimerization.
    • Allows interaction with the IkBs.
    • DNA Binding.
    • Contains the NLS.
picture of rel proteins
Picture of Rel-Proteins
  • View of NF-kB binding DNA.

NF-kB

DNA

transcriptional regulation by nf b mechanisms
Transcriptional Regulation by NF-B -- Mechanisms
  • Binds to a unique sequence found in the kB-responsive 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.
    • 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
Specificity- Transcription Factor

Are there other mechanisms of specificity?????????

From Science, 2004, 306:632-635

reminder how a tf works
Reminder: How a TF works

http://life.nthu.edu.tw/~lslpc/StrucBio/chapter9/chapter9_2.html

reminder how a tf works16

Enhancer

IID

Initiator

TATA BOX

-25

Reminder: How a TF works

NF-B

Diagram based on and adapted from Struhl, K., Cell 84: 179-182

genes regulated by nf b good vs bad
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 k b really important
Is NF-kB really Important????
  • Through the use of Knock-Out animals the critical role NF-kB 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 k b activation
Mechanism: NF-kB Activation
  • NF-B activity is regulated by a family of inhibitors termed IB which include IB, IB, IB, the p105 and p100 precursors, and Bcl-3.
  • Specifically, the IBs 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 IB allowing it to translocate to the nucleus and transactivate B-responsive elements.
the i k bs
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 k b and i k b family21
The NF-kB and IkB Family
  • Schematic of the NF-kB/IkB families
  • Details KEY features

DeMeritt & Yurochko; In, Recent Res. Devel. Virol., Vol. 7, pp. 55-107.

i k b vs i k b
IkB vs. IkB
  • 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.
i k b vs i k b23
IkB vs. IkB
  • IkB is a 46 kDa protein.
  • First discovered in 1995.
  • Blocks the NLS of NF-kB.
  • Regulates the persistent release of NF-kB.
  • Also appears to protect NF-kB from the negative effects of IkB
  • Specificty????
mechanism i k b regulation
Mechanism: IkB Regulation
  • The IkBs contain critical serine residues.
    • 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.
i k b degradation
IkB Degradation
  • Schematic of the regulatory serines and a quick look at the ubiquitination event (occurs at lysines 21 and 22 on IkB).
    • E1 - ubiquitin-activating enzyme
    • E2 - ubiquitin-conjugating enzyme
    • E3 - ubiquitin-ligating enzyme
is i k b really important
Is IkB really Important????
  • IkB KO -- born normally but die of a wasting disease by day 7.
the ikks
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, IKKa and IKKb are catlytic subunits (85 & 87 kDa, respectively).
  • The third member, IKKg (NEMO), is a regulatory subunit (48 kDa).
the ikks28
The IKKs
  • IKKa and IKK 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 IKKa and IKK. Probably interacts with IKK and IKK as a dimer or a trimer.
schematic of the ikks
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
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
Are the IKKs really Important?
  • In Mice:
    • 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).
    • IKK KO -- embryonic lethality (similar to the p65 & IKK KO animal).
  • Suggests what???????????
defects in ikk a kos
Defects in IKKa KOs

Hu et al., 1999, Science 284:316-320

more defects in ikk a kos
More Defects in IKKa KOs

Hu et al., 1999, Science 284:316-320

defects in ikk kos
Defects in IKK KOs

Li et al., 1999, Science 284:321-325

are the ikks really important35
Are the IKKs really Important?
  • In humans, there is a diagnosed genetic defect in which IKKg 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
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 g nemo another role
IKKg/NEMO - Another Role
  • IKKg or NEMO can function as a bridge to the interferon signaling pathway
  • Thus IKKg also has the capacity to regukate signal transduction pathways independent of its role in the regulation of NF-B activation
other upstream regulators
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-kB Inducing Kinase)
    • MEKK1 (A MAP3K)
    • Ras/Raf
    • Others
more details possible specificity
More Details Possible Specificity????
  • NIK seems to preferentially activate IKKa.
  • MEKK1 seems to preferentially activate IKKb.
  • Suggests what????
what activates nf k b
What Activates NF-kB?????
  • Cytokines
  • Growth Factors
  • Cell Adhesion
  • Viral Infection
  • Thus a Receptor-Ligand mediated event.
one last concluding figure
One Last Concluding Figure

Molecular Cell Biology; 4th Edition

Other mechanisms of specificity?????

everything you ever learned in one cartoon
Everything you ever learned in one cartoon!

IMAGES FROM: G. Orphnides and D. Reinberg 2002, Cell 108: 439-451

nf k b biological implications health human disease
NF-kB Biological Implications:Health & Human Disease
  • NF-kB regulation is essential to many aspects of our health including:
    • cellular development
    • cellular survival
    • the immune system
  • Diseases associated with a dysregulation of NF-kB.
    • 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 k b biological implications health46
NF-kB Biological Implications:Health
  • NF-kB regulation is essential to many aspects of our health including:
    • cellular development
    • cellular survival
    • the immune system