Regulation of IKK - Transcription Factors NF-kappaB - page 29

B activation by most proinflammatory
stimuli [32,33]. In contrast, an intact activation loop in IKK

phosphorylation of I κ B proteins and NF- κ

α is required for acti-

vation of the alternative NF- κ B pathway, which leads to processing to p100 (see

Section 3.4.1.2 ) . The HLH motif has also been reported to be involved in regulation

of IKK activity. Mutations within this motif decreased IKK activity, both in tran-
siently transfected cells and with purified proteins, [15,16] although HLH-deficient
mutants still dimerize via their LZ domains and bind to NEMO. Deletion of a C-
terminal fragment of IKK

encompassing the HLH-motif and the NBD destroy
kinase activity [30]. Interestingly, the activity of such a C-terminal deletion mutant
could be restored by coexpression of separate C-terminal HLH- and NBD-containing
fragments [30]. As such, the HLH-motif physically interacts with the kinase domain
and seems to serve as an endogenous activator of IKK in a manner similar to the
function of the cyclin subunits of cyclin-dependent kinases (CDKs) [34]. C-terminal
of the HLH motif and prior to the NBD, IKK

β

contain a stretch of serines, which
are heavily phosphorylated during IKK activation [30]. Phosphorylation at these
sites requires IKK activity, implicating autophosphorylation as underlying mecha-
nism. In contrast to phosphorylation of the activation loop however, phosphorylation
of the C-terminal cluster has a negative autoregulatory function [30]. Replacement
of 10 of the C-terminal serines with alanines significantly prolongs TNF

α / β

α -induced

IKK activity, whereas replacement of these serines with (phosphomimetic)

glutamate residues reduces IKK activity [30]. Given the transient nature of IKK
activation by most stimuli investigated so far, this autophosphorylation may be a
primary mechanism for negative regulation of IKK activity, followed by phos-
phatase-dependent dephosphorylation of the activation loop, reverting IKK activity

β

to its baseline levels ( Figure 3.1b ). Protein phosphatase 2C β has been found to

associate with IKK and reduce its phosphorylation and activity when overexpressed

in HEK293 cells [35]. In accordance with these findings, small interfering RNA
(siRNA)-based knockdown of PP2C

β

β leads to prolonged TNF α -induced IKK activ-

ity [35]. Whether PP2C β specifically dephosphorylates the serine residues in the

activation loop of IKK needs to be investigated.

By definition, the alternative NF-

β

and
NEMO. Only a subset of TNF receptor family members, such as BAFF-R and CD40
on B cells and LT

κ B pathway does not depend on IKK β

R in splenic stromal cells, activate this pathway [8]. There is still
much less information about the molecular events involved in IKK

β

α -dependent

processing of p100 compared to IKK B phosphorylation. Indeed, most

of our knowledge on the function of this pathway is based on genetic rather than
biochemical analysis. IKK

β -dependent I κ

-AA mutant
are deficient in p100 processing [36]. Overexpression of a constitutively active form
of IKK

α -deficient cells or cells expressing an IKK α

, induces p100 processing in HEK293
cells [36]. A C-terminal serine residue in p100, which functionally seems to corre-
spond to serines 32 and 36 in I

α , but not catalytically inactive IKK α

κ B α , is required for IKK α -dependent processing and

is phosphorylated by purified IKK α in vitro [36]. Surprisingly, stimulation-depen-

dent activation of IKK α dimers has not been clearly demonstrated. Whether this is

due to a low-level IKK activation, reflected by the slow kinetics of p100 processing

or due to other factors, such as controlled compartmentalization of the kinase and
its substrate, is unknown. The inability to show regulated activation of IKK

α

α dimers