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  IKKs  Upstream Regulators

NF-

k

B Biological Implications: Human Disease

 Diseases associated with a dysregulation of NF k B.

 Atherosclerosis  Asthma  Arthritis  Cancer  Diabetes  Inflammatory bowl disease  Stroke  Viral Infections (AIDS)

NF-

k

B Biological Implications: Health

 NF k B regulation is essential to many aspects of our health including:  cellular development  cellular survival  the immune system

What Are We Talking About??

Simple (?) Model Of The Known Players In NF-

k

B Induction

 Quick Overview of the NF k B/I k B Signaling Pathway

IL-1 IL-1R1 Virus TNF TNFR1 PMA Other Ras IRAK ?

TRADD Cell Memb.

PKC TRAF2 TRAF6 NIK MEKK1 MAPK IKK (



and



) pp90rsk I

k

B Phosphorylation NF-

k

B/I

k

B Free NF-

k

B NF-

k

B Translocation NF-

k

B Responsive Genes I

k

B Degradation Nuclear Memb.

The Players

 NF k B  The I k Bs  The IKKs  Other Upstream Regulators

History of NF-

k

B

 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

    NF k B is a heterodimeric transcription factor from the

rel

-family of transcription factors. Classic NF k 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

 Schematic of the NF k B/I k B families  Details KEY features QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this pi cture.

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

NF k 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 k B subunits contains a

rel

homology domain.

RHD - Defines this Family

 Common to all members of the NF k B family.  Is ~ 300 a.a. domain.

 Is a multifunctional domain.

 Controls NF k B dimerization.

 Allows interaction with the IkBs.

 DNA Binding.

 Contains the NLS.

Picture of

Rel

-Proteins

 View of NF k B binding DNA.

NF-

k

B DNA

Transcriptional Regulation by NF k B -- Mechanisms    Binds to a unique sequence found in the k B responsive promoters (5’-GGGRNNYYCC-3’). c-Rel, RelB, and RelA (p65) contain transactivation domains.

The NF k B family members interact with other transcription factors and members of the basal transcriptional machinery.

 NF k 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

Enhancer NF k B IID TATA BOX -25

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

Initiator

Genes Regulated by NF k B Good 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????

 Through the use of Knock-Out animals the critical role NF k 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-

k

B Activation

  NF k B activity is regulated by a family of inhibitors termed I k B which include I k B  , I k B  , I k B  , the p105 and p100 precursors, and Bcl-3. Specifically, the I k Bs binds to NF k 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 k B from I k B allowing it to translocate to the nucleus and transactivate k B responsive elements.

The I

k

Bs

  There are two main I k Bs   I k B  I k B  There are also other less studied I k Bs or I k B like molecules.

 I k B   The C-terminal portions of p100 and p105.

 BCL3

The NF-

k

B and I

k

B Family

 Schematic of the NF k B/I k B families  Details KEY features QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this pi cture.

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

I

k

B



vs. I

k

B

  I k B  is the prototypic I k B.

 We first discovered it in 1990.

   It is a 37 kDa protein.

Binds to NF k B and blocks its NLS.

Regulates the rapid release of NF k B and its rapid down regulation.

 Also contains a nuclear export signal which is important in the removal of NF k B from the nucleus.

I

k

B



vs. I

k

B

  I k B  is a 46 kDa protein.

 First discovered in 1995.

 Blocks the NLS of NF k B.

  Regulates the persistent release of NF k B.

Also appears to protect NF k B from the negative effects of I k B   Specificty????

Mechanism: I

k

B Regulation

 The I k Bs contain critical serine residues.

  I k B  I k B  -- S32 & S36 -- S19 & S23   These serines are the targets of upstream serine kinases termed IKKs (I k B Kinases).

Following phosphorylation, the I k Bs are ubiquitinated and targeted for degradation by the 26S proteasome.

I

k

B Degradation

 Schematic of the regulatory serines and a quick look at the ubiquitination event (occurs at lysines 21 and 22 on I k B  ).

 E1 - ubiquitin-activating enzyme   E2 - ubiquitin-conjugating enzyme E3 - ubiquitin-ligating enzyme

Is I

k

B really Important????

 I k B  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, IKK g (NEMO), is a regulatory subunit (48 kDa).

The IKKs

 IKK  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 g 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

 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:   IKK  KO -- born alive but died shortly after birth. Showed severe muscular and skeletal defects. Had normal activation of NF k B following proinflammatory stimuli.

IKK  KO -- embryonic lethality (similar to the p65 KO  animal). IKK g KO -- embryonic lethality (similar to the p65 & IKK  animal).

KO  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?

 In humans, there is a diagnosed genetic defect in which IKK g 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

g

/NEMO - Another Role

 IKK g or NEMO can function as a bridge to the interferon signaling pathway  Thus IKK g also has the capacity to regukate signal transduction pathways independent of its role in the regulation of NF k 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 k B 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-

k

B?????

 Cytokines  Growth Factors  Cell Adhesion  Viral Infection  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-

k

B Biological Implications: Health & Human Disease

  NF k 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 k 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-

k

B Biological Implications: Health

 NF k B regulation is essential to many aspects of our health including:  cellular development  cellular survival  the immune system