Biomedical Engineering Reference
In-Depth Information
was identified by sequence homol-
ogy comparisons and site direction mutagenesis [13,15]. These studies characterized
a conserved N-terminal sequence containing two serines, which were phosphorylated
after phorbol ester stimulation, that are required for signal-induced I
The motif regulating the degradation of I
κ
B
α
ubiquiti-
nation and degradation by the proteasome. This information was critical for the
subsequent molecular characterization of both the I
κ
B
α
κ
B kinase (IKK) complex (see
Chapter 3
of this topic) and the I
κ
B ubiquitin ligase. Using a set of specific phos-
phopeptides, Yaron and colleagues showed that the phosphorylation-based motif
(DpSGXXpS) of I
B recognition by components of the ubiq-
uitin-system
in vitro
[16]. Mass spectroscopy identified
κ
B
α
is sufficient for I
κ
β
TrCP as a protein that could
specifically interact with the I
κ
B phosphopeptide motif [17]. Significantly, a
Droso-
phila
homolog of
TrCP,
slimb
, had previously been identified by Jiang and Struhl
in a genetic screen as a likely E3 for
β
-catenin and Cubitus Interruptus (Ci) [18].
Several groups [19,20,21] then demonstrated that
β
TrCP is the substrate binding
(receptor) subunit of an Skp1 cullin F-box (SCF)-type E3 ligase [22]. The structural
basis for recognition of phospho-I
β
B E3 ligase was established by
Pavletich and his colleagues, who solved the crystal structure of the substrate inter-
acting pocket of
κ
B
α
by the I
κ
β
TrCP in complex with the cognate phosphopeptide from
β
-catenin
[23]. The role of
β
TrCP in NF-
κ
B activation is not limited to controlling I
κ
B
α
degradation, and
β
TrCP was subsequently shown to control the proteasome-mediated
degradation of I
κ
B
β
, I
κ
B
ε
[24,25], and NF-
κ
B1 p105 [26,27,28], as well as the
signal-induced processing of NF-
B2 p100 to p52 by the proteasome [29].
Recently, ubiquitination has been shown to have a novel role in NF-
κ
B activation,
which does not involve proteasome-mediated proteolysis of target proteins. Exper-
iments by Chen and colleagues used
in vitro
reconstitution assays to investigate how
IKK is activated [30] and discovered that the attachment of a unique type of poly-
ubiquitin chain linked through K63 of ubiquitin to target proteins is an essential step
in cytokine activation of IKK [31]. Research into NF-
κ
B regulation therefore con-
tinues to reveal novel mechanisms by which ubiquitin regulates intracellular signal-
ing, which are likely to have far-reaching implications in our understanding of the
control of cell physiology.
κ
4.2
SIGNAL-INDUCED I
κ
B PROTEOLYSIS
The majority of stimuli that induce NF-
κ
B activation target all three I
κ
Bs (I
κ
B
α
,
I
κ
B
β
, I
κ
B
ε
) for phosphorylation by the IKK complex (see
Figure 4.2
)
[32]. IKK-
mediated phosphorylation promotes I
κ
B ubiquitination and subsequent degradation
by the proteasome [33]. I
κ
B
α
has been shown to be a better substrate for IKK when
complexed with NF-
κ
B dimers, providing a mechanism to ensure that NF-
κ
B-free
I
is protected from fortuitous, IKK-induced ubiquitination and degradation [33].
Mutation of the IKK phosphorylation sites (S
32
or S
36
of I
κ
B
α
κ
B
α
, or the equivalent
sites of the other I
κ
Bs) renders I
κ
Bs resistant to signal-induced degradation and
expression of such mutants of I
κ
B
α
has been used to block NF-
κ
B both in cell lines
and
in vivo
.
Following its phosphorylation by IKK, I
κ
B
α
is recognized by the F-box-WD
repeat protein
β
TrCP (also called E3RS
IκB
or Fbw1a) — the receptor subunit of the
