NF-kB

 

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Nuclear Factor necessary for Ig kappa (k) light chain transcription in B cells.

 

Referred to a the prototype of dimeric transcription factors

 

OVERVIEW

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


(adapted  from Baeurele et al., 1996, Cell 87, 13-20)

 

NF-kB exists as a dimer in the cytoplasm – complexed to an inhibitor protein
IkB that prevents trafficking to the nucleus.
 
NF-kB activation is associated with the release of IkB – allowing nuclear targeting.

 

 

        NF-kB forms

 

·       p50 (NF-kB1)

·       p52 (NF-kB2)

·       p65 (RelA)

·       cRel

·       RelB

 

 

STRUCTURE

 

Related to the proto-oncogene , c-Rel. 

 

 “Rel”  proteins were first characterized as cryptic DNA binding proteins

 

Formed from two DNA binding subunits made up from  homo- or heterodimers 

“rel” motif proteins. This is approximately 300a.a. 

 

The Rel area contains  

 

·       Dimerisation motifs

·       DNA binding region

·       A nuclear localisation signal. 

·       Interaction with the inhibitor protein - IkB (see later notes).

 

The C –terminus (not found in p50 or p52) contains varies greatly in each rel

protein and is responsible for transactivation.

 

 Click for larger image

 

 

Interaction with DNA

 

     Determined for the human and mouse p50:p50 homodimer

–       (see Ghosh et al., 1995, Nature, 373 : 303-310 in General Office Papers)

–       though  the  most common interaction is p50/p65.

 

·       Resembles a “butterfly”- with two pairs of wings connected to a central core of DNA.

 

·       The Rel region folds into two domains- connected by a 10 residue linker (238-247aa)

 

·       C-terminal domain (248-350aa) – core b-sheets – similar in structure to the immuno-

·       globin family.

 

·       This region is responsible for dimer formation- which brings together 10 loops (five from

·       each subunit) – to fill the major groove of a DNA molecule and generate a sequence

·       specific DNA-binding surface.

 

·       Effectively wraps around the DNA molecule.

 

·       Binding is augmented by the a-helix in the N-terminal region which bind to the minor grove.

 

·       Any variation in this dimer/loop interface will affect NF-kB specificity


Genes regulated by NF-kB have “ kB” sites of the following consensus DNA sequence

 

5’ GGGRNNYYCC 3’.             R = Purine, Y = Pyrimidine, N = any base.

 

(the variation reflects the varying combinations of homo and heterodimers

          that can form.)

 

The sequence results in an unusual conformation of DNA-

 

One turn of DNA molecule usually consists of 20bp but is 10.7bp in this region-

This tight twist results in a deep major groove.

 

 

The Interaction with IkB

 

(Ghosh et al., 1995, Nature, 373 : 303-310)

 

Nuclear localization signal is found at residues 360-364 on NF-kB, which is masked by IkB.

 

Found just above the dimer interface- in the flexible linker domain. - therefore the interaction

with IkB is likely to alter the “butterfly” configuration.

 

 

 

 

I-kB: The NF-kB sequestrator

 

Bind to rel region of NF-kB to block the nuclear localization signal so that the

 transcription factor remains in the cytoplasm.

 

 

 

IkB inhibitors all have 5-7 ankyrin (30aa) repeats domains

 

Release

 

·       Involves the phosphorylation of serines 30 + 32 (N-terminus).

 

·       Followed by ubiqutinisation of lysines 21 and 22 and degradation by the 26S

proteosome. C-terminus is essential for the interaction with the “proteosome”

–       as part of ubiquitin – mediated degradation. 

 

What is ubiqutinisation?

 

Protein-degradation service – proteins are targeted for proteolytic digestion by

being decorated with ubiquitin

 

·       E2 “ubiquitin conjugating enzyme”. This may directly transfer ubiquitin to

lysines on the target protein  followed by poly-ubiquitinisation.  Further ubiquitin

proteins are added to lysines lying within the conjugated ubiquitin protein.

 

·       Proteosome  -the polyubiqutin chain targets the protein for degradation.

 

Click for more information on ubiquitinization

 

Suppressing IkB degradation: Protection using SUMO-1.

 

SUMO-1 binds to the same lysines used for ubiquitinisation. This creates a “privileged

 pool of IkB” that doesn’t respond to cytokine signals. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Forms of IkB

 

Four main forms of IkB with differing affinities for NF-kB forms - suggesting the

possibility of selective degradation to release certain NF-kB forms.

 

IkB-a: 37kDa protein. The “classical IkB” in terms of regulation. 

 

IkB-b: 45kDa protein. With IkBa is responsible for inhibiting the majority of p50:p65.

 

IkB-e: Only associated with RelA  and c-Rel – indeed relA is seldom complexed

with other forms. Only slowly degraded following stimulation. Regulates specific

 genes e.g. IL-8.

 

IkB-g: 70 kDa protein only detected in lymphoid cells. Identical to the c-terminal

regions of p105. Probably only inhibits p50 and p65 homodimer.

 

Viruses: African swine fever virus – produces an IkB –like protein (A2302) which

can suppress NF-kB activation and therefore the inflammatory response.

 

A REGULATED NEGATIVE FEEDBACK LOOP

 

·       NF-kB induces the expression of many of suppessors (IkB).

·       IkB have nuclear localization signals  -so can remove NF-kB from kB sites and suppress expression.

·       Cells must avoid NF-kB effects being transitory but retain this as a potential negative feedback mechanism

·       This is done to a great degree IkB-b.

·       Both phosphorylated and non-phosphorylated forms of IkBb bind to NF-kB but only the

phosphorylated form can prevent DNA binding ie. remove the transcription factor.

 

 

          STEP1 Thus, IkB-a is ubiquitinated and degraded –

          STEP2. NF-kB moves to the nucleus

          STEP3 Can bind to IkB-a promoter which has NLS which can remove NF-kB from kB sites

          STEP4 BUT this can be blocked if NF-kB is bound by non-phosphorylated IkB-b -( Persistent activation)

          STEP5. Phosphorylation of IkB-b will lead to this removing NF-kB from sites (Suppression of gene expression)

 

 


Biogenesis of NF-kB - p50/p52.

 

p50 and p52 are synthesized from the N-terminus of larger precursors p105 and p100

 respectively.

 

At C-terminus – “ankyrin repeats” – these allow protein / protein interactions.

 
These can interact with the NLS to suppress nuclear trafficking –as does IkB

 

p50/p52 proteolytic cleavage is ATP dependent and involves ubiquitinisation

 

Cleavage of the ankyrin repeat regions immediately results in nuclear trafficking.


NF-kB multi-tasking

 

The variable interactions between NF-kB monomers with varying forms of IkB allow a similar

mechanism to be used for very differing effects.

 

Evidence: -

 

(i) Knock-outs

 

Rel A-/-                        Causes apoptosis of liver cells in mouse prior to birth

                             (i.e. primarily anti-apoptotic)

 

Rel B-/-                  Lethal multiple organ inflammation.

                             (i.e. primarily anti-inflammatory)

 

c-Rel-/-                   B and T cell deficiencies

 

p52-/-                     Altered lymph mode architecture

 

 

(ii) “Artificial” formation of dimers has different effects.

 

p50/c-Rel, p65 (Rel-A) /p65 (Rel-A), p65 (Rel-A)/c-Rel dimers will activate transcription

 

p50 & p52 homodimers will suppress transcription.

 

How do cells influence what dimers will be form/ be activated?

 

(i) Tissue specific expression of subunits . This influences which homo-/heterodimer will

     predominate.

 

(ii) Variable interactions with IkB - forms

 

(iii) Variable upstream activation – influencing the   phosphorylational changes