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Nonetheless, MEKK3-deficient fibroblasts exhibit defects in IL-1 and TNF
-
induced IKK (and JNK1/2) activation, a phenotype similar to RIP1-deficient cells
[47,48,58]. MEKK3 has been shown to interact with RIP1 and overexpressed
MEKK3 can still activate NF-
α
B and JNK in RIP- and TRAF2-deficient cells,
indicating that MEKK3 cooperates in the RIP1-dependent pathway downstream of
TRAF2 and RIP1 [58]. Interestingly, when RIP-deficient elongation factor (EF) cells
were reconstituted with a MEKK3 fusion protein containing MEKK3 and the death
domain (DD) of RIP1, TNF
κ
B activation was restored, indicating
that RIP1 might serve as an adaptor protein that recruits MEKK3 into a TNFRI-
induced signaling complex [63]. The NF-
α
-induced NF-
κ
B inducing activity of the MEKK3-RIP1-
fusion protein depended on an intact ATP-binding pocket of MEKK3, implying that
MEKK3 catalytic activity is required for IKK activation [63]. Although it is formerly
possible that binding and oligomerization of the DD of RIP linked to MEKK3 creates
a nonphysiological situation, thereby activating MEKK3, an attractive scenario
would be that TRADD oligomerization induces recruitment of TRAF2 and RIP1
through direct protein-protein interactions. TRAF2 (together with TRAF5) recruits
the IKK complex through binding of its IKK
κ
subunits, which may be further
supported via interaction between RIP1 and NEMO. RIP1 serves as adaptor that
brings MEKK3 into close proximity with the IKK complex, allowing IKK activation,
α/β
possibly through direct phosphorylation of its catalytic subunits (
Figure 3.2a
).
It
needs to be stressed, however, that direct phosphorylation of IKK
by MEKK3
has not been demonstrated; therefore, it remains to be established whether MEKK3
acts at all as an IKK-K
in vivo
. TAK1 was originally identified as a kinase involved
in TGF
α/β
signaling [64]. Later, it was found to be activated in response to other
stimuli, such as TNF
β
and IL-1 [65,66]. It was also identified as component of an
IKK activating complex, coeluting with IKK-inducing activity in an
in vitro
, cell-
free reconstitution system [55]. In contrast to MEKK3, TAK1 does not activate
NF-
α
B when overexpressed alone, but depends on coexpression of other molecules,
that is, TAB1, TAB2, or TAB3 [55-57,67,68].
However, the analysis of TAB1 and TAB2-knockout mice failed to reveal a role
for these molecules in IKK activation. Neither single knockout exhibited defects in
TNF
κ
B activation [60,68
]
. The results indicate instead that
TAB1 is involved in the TGF
α
- or IL-1 induced NF-
κ
-pathway [69]. However, RNAi knockdown experi-
ments have shown that knockdown of TAB2 or TAB3 alone had no effect on NF-
β
B
activation, but the simultaneous knockdown of both molecules resulted in a signif-
icant signaling defect [67]. Given the structural and functional similarities between
these proteins, it is possible that TAB2 and TAB3 compensate for each other. Both
proteins were found to be recruited to TAK1 during TNF
κ
and IL-1 signaling, and
also to interact with TRAF2 and TRAF6, which are involved in TNF
α
and IL-1
signal transduction, respectively [67]. Interestingly, the C-termini of TAB2/3, which
were demonstrated to be required for their function contain a ZnF motif that is
typical for ubiquitin-binding proteins [71]. Notably, the TRAF proteins contain
RING-finger domains, which may be involved in assembly of K63-linked polyubiq-
uitin chains [72]. Moreover, activation of cells by TNF
α
and IL-1 induces TRAF2-
dependent ubiquitination of RIP1 [52,73]. Further support for atypical (K63-medi-
ated) ubiquitination as an important signaling event comes from the observation that
α
