Regulation of IKK - Transcription Factors NF-kappaB - page 28

covering the NBD of IKK β , which has higher affinity to NEMO than the corre-

sponding part of IKK α , prevents IKK activation by tumor nuclear factor α (TNF α)

[21]. Likewise, deletion of NEMO prevents IKK and NF- B activation by multiple

stimuli [22,23]. These data illustrate that activation of IKK by upstream pathways
require NEMO. However, our understanding regarding the molecular mechanism of
NEMO function and relation to upstream activators is far from being complete.
Importantly, some data clearly demonstrate that different stimuli activate NEMO by
different molecular mechanisms. For example, the zinc finger motif at the very C-
terminus of NEMO is essential for IKK activation by ionizing irradiation but seems
to be dispensable for LPS-induced IKK activation [22,24,25]. The mechanisms of
IKK activation by distinct stimuli will be discussed in Section 3.4 .
Although formation of a catalytically active protein complex of about the size
of the endogenous IKK complex with recombinant IKK

κ

and NEMO suggests
that the core-IKK complex is indeed made up of these three subunits, other proteins
have been described to associate with IKK in vivo , for instance, Hsp90 [26]. How-
ever, Hsp90 is a chaperon protein that interacts with many protein kinases [27] and
likely is not specific to IKK.
IKK

α / β

contain several features that distinguish them from other kinases,
such as LZ and HLH motifs. The LZ domain mediates dimerization of the kinases
and is essential for kinase activity [15,16,28]. The kinase domain of IKK

α and β

is
similar to that of other serine-threonine kinases, containing a highly conserved
adenosine triphosphate (ATP)-binding site. Mutations of the conserved lysine in this
region (K44) generate catalytically inactive mutants of IKK

α/β

. In vitro
kinase assays using reconstituted complexes showed that homodimers containing
two defective subunits were catalytically inactive, while heterodimers that contain
a single active subunit still exhibit kinase activity [16]. Accordingly, transfection of
limiting amounts of epitope-tagged IKK

α and IKK β

(K44)-mutants can be used to immu-
nopurify catalytically active endogenous IKK

α/β

α or IKK β from cells, but overexpres-

sion of catalytically inactive IKK B activation

by different stimuli, probably by competition with endogenous active forms of the
kinases [15,28,29].
There is considerable evidence that both IKK

α or IKK β can inhibit IKK and NF- κ

need to be phospho-
rylated to become activated. Purified IKK is inactivated upon incubation with protein
phosphatase 2A (PP2A), whereas treatment of cells with the PP2A inhibitor ocadaic
acid results in IKK activation [9]. Like other protein kinases, the kinase domains of
IKK

α and IKK β

contain an activation loop, which is subject to phosphorylation at
two serine residues that induces a conformational change leading to kinase activation

α and IKK β

( Figure 3.1b ) [29-31]. Replacement of those two serines in IKK β (S177/S181) with

alanines (IKK -AA) prevents kinase activity, while replacement with phosphomi-

metic glutamates results in a constitutively active kinase [30]. Both of these serines
are phosphorylated in vivo in response to proinflammatory stimuli, such as TNF

β

α

and IL-1 [30]. Substitution of wt IKK β with IKK β -AA also prevents IKK and NF- κ B

activation in response to TNF α or IL-1, similar to the situation in IKK β -deficient

cells [28,30]. The activation loop of IKK and also

becomes phosphorylated at the corresponding serines during cell stimulation [30].
However, IKK

α is identical in sequence to IKK β

α phosphorylation is not critical for activation of the IKK complex,