Research Report 2000 - MDC

Regulation of
Transcription in
Mammalian Cells
Claus Scheidereit
Cellular growth and differentiation
largely depends on the expression of
sets of genes which are controlled
essentially at the level of
transcription. Differential gene
expression patterns are programmed
by transcription factors, whose
activity in turn is modulated by
complex networks of signal
transduction cascades. The main
objective of our laboratory is to
understand how signal transduction
processes are coupled to transcription.
A model system with wide
physiological and medical relevance is
nuclear factor kappaB (NF-κB) and its
co-regulators and accessory proteins.
A major goal of our research is to
understand the structures and
mechanisms underlying gene
regulation by this complex system.
Pathways and structures that
regulate NF-κB activity
The pleiotropic transcription regulator
nuclear factor κB (NF-κB) plays an
important role in the inducible
expression of a large number of genes
which encode cytokines, surface
receptors, adhesion molecules,
transcription factors and other
molecules controlling various immune
functions as well as cellular growth or
programmed cell death. In its inactive,
latent form NF-κB is kept in the
cytoplasm by association with IκB
molecules, which inhibit nuclear
translocation and DNA binding of NFκB.
Stimulation of cells with a variety
of agents, such as bacterial
lipopolysaccharides (LPS), phorbol
esters (PMA), tumor necrosis factor α
(TNFα), interleukin-1 (IL-1) or UV
light results in the proteolysis of the
IκB molecules and liberation of active
NF-κB into the nucleus. Induced IκB
proteolysis is triggered by IκB
phosphorylation mediated by an IκB
kinase (IKK) complex, which is
activated by the many NF-κBstimulating
pathways. The
composition and regulation of the
IKK complex is under investigation
and, of particular interest, is the
identification of molecules which
directly activate or inhibit the
complex.
Differential regulation of NF-κB
activity by IκBα, IκBβ, p105 and
Bcl-3
The mammalian NF-κB family
consists of five members, p50, p65,
p52, the protooncogene product c-Rel
and RelB. These conserved proteins
form various hetero- and homodimers
and are bound by IκB molecules
IκBα, β, and ε, the IκB-like precursor
proteins for p50 and p52, p105 and
p100, respectively, or by the nuclear
IκB homologue Bcl-3. We have found
that in human cells IκBβ is expressed
as two distinct splicing variants,
IκBβ1 and IκBβ2. While both forms
equally well associate with NF-κB,
they differ in their responsiveness to
signals and sub-cellular localization.
Due to a lack of components of a
carboxyterminal PEST sequence,
IκBβ2 is only weakly degraded in
response to inducing agents and so its
relative abundance determines the
responsiveness of a given cell. In B
lymphocytes, IκBβ1, but not IκBβ2,
is found in the nucleus and may
contribute to the persistent NF-κB
activity in these cells. These findings
may also indicate that the efficiency
of the IKK complex, which
phosphorylates IκBβ1 and 2 at
invariant aminoterminal residues, is
affected by the presence of the PEST
domain. The IKK complex
phosphorylates IκBβ and IκBα at a
conserved signal response domain and
this sequence, containing also lysines
for phosphorylation-dependent
ubiquitin-conjugation, is sufficient to
confer inducible degradation. A short
50 amino acid sequence of IκBα,
when fused to other proteins, triggers
degradation of these proteins when
cells are activated by TNFα or other
agents which activate IKKs. We have
also found that the NF-κB precursor
proteins, p105 and p100, which on
processing give rise to p50 and p52,
sequester other NF-κB subunits
including their processing products in
the cytoplasm and so act like IκB
molecules. On stimulation with NFκB
activating agents, cellular p105 is
phosphorylated by same kinetics as
IκBα. We have now found that IKKs
phosphorylate p105 and that the major
sites are three serines close to the
carboxyterminal end of p105.
Phosphorylation at these sites by
IKKs results in rapid, complete
degradation of p105 by the
proteasome. p105-associated NF-κB
subunits, such as p50, which is
formed by processing of p105, are
liberated and are transported to the
nucleus. Thus, in parallel with the
release of NF-κB dimers by induced
degradation of IκBα or IκBβ1, other
NF-κB subunits, including p50
homodimers, are released by p105
degradation. Since p50 homodimers
are specifically bound by the nuclear
IκB homologue Bcl-3, TNFα or IL-1
stimulation results in rapid
accumulation of p50-Bcl-3 complexes
in the nucleus. The protooncogene
product Bcl-3 acts like a
transcriptional co-activator for p50
homodimers, which lack their own
transactivation domains. Several
nuclear cofactors and chromatinmodifying
proteins have been
identified which potentiate the
transcription activation potential of
Bcl-3-p50 complexes. These
accessory proteins include the histone
acetylase, Tip60, which
superstimulates Bcl-3-mediated
transcriptional activation and forms
quarternary complexes with p50-Bcl-3
bound to promoter DNA.
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