Cell Biology - ModelsThe NF-B/IB 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-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?

The PlayersNF-BThe IBsThe IKKsOther Upstream Regulators

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.

Picture of Rel-Proteins View of NF-B

binding DNA.

DNA

NF-B

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

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

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

Is IB really Important???? IBα KO -- born normally but die of a wasting

disease by day 7.

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???????????

Defects in IKKα KOs

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

More Defects in IKKα KOs

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

Defects in IKKβ KOs

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

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)

Affected Males

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

At this point, what do we know?

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.

One Last Concluding Figure

Other mechanisms of specificity?????

Molecular Cell Biology; 4th Edition

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.

NF-B Biological Implications:HealthNF-B regulation is essential to many

aspects of our health including: cellular development cellular survival the immune system