T-cell costimulation by simultaneously triggering of the T-cell receptor (TCR) and the auxiliary receptor CD28 leads to the synergistic activation of JNK (63
) and NF-κB (20
). Here we show that the serine/threonine kinase MLK3 plays an important role in the T-cell costimulation-induced activation of NF-κB. Since MLK3 is known to activate the JNK pathway (65
), this finding contributes to our understanding of how a given stimulus can simultaneously activate JNK as well as NF-κB. The behavior of MLK3 as a dual activator of JNK and NF-κB is reminiscent of MEKK1, which activates JNK via phosphorylation of JNKK1 (MKK4/SEK) (42
) and triggers NF-κB by direct phosphorylation of both IKKs (38
). In the absence of specificity constants of MLK3 for its substrates JNKK1 (66
) and IKKα and IKKβ (this study), it is impossible to estimate whether MLK3 has a bias for either of the two pathways. The activation of a given pathway by more than one upstream activator is not without precedent. An example is provided by MEKK1-deficient embryonic stem cells, which are unable to activate JNK upon exposure to cold stress and microtubule disruption but display normal JNK activation in response to heat shock, UV irradiation, and anisomycin (72
). The maintenance of JNK activation in response to certain stimuli points to the compensatory action of other JNKK kinases such as MLK3.
Here we identify T-cell costimulation as a physiological inducer of MLK3 activity. We suggest a scenario in which activated MLK3 promotes IL-2 transcription in two ways: first, it contributes to the activation of NF-κB, a transcription factor transactivating from the NF-κB binding site and the CD28RE/AP element contained in the IL-2 promoter. Second, triggering of MLK3 results in the activation of JNK, a kinase that is necessary for transactivation from the CD28RE/AP element (34
) and the AP-1 sites (31
) contained within the regulatory region upstream from the IL-2 gene. The T-cell costimulation-induced NF-κB-dependent transcription could not be absolutely blocked by dominant negative MLK3, raising the possibility for the existence of further kinases feeding costimulatory signals into the IKC. A candidate is the MAPKKK Cot, which was recently shown to participate in TCR-CD28-induced NF-κB activation (43
). However, the pathway triggered by Cot is distinct from that activated by MLK3. Whereas Cot leads to the activation of NIK (43
), MLK3 does not act upstream from NIK and directly phosphorylates both IKKs. Since the MEKK1-homologous proteins MEKK2 and MEKK3 were recently identified as NF-κB activators (74
), it may well be that MLK3- and Cot-homologous proteins also contribute to the TCR-CD28-mediated activation of NF-κB. However, it remains to be seen whether endogenous Cot and MEKK1 are activated by T-cell costimulation.
This study identifies MLK3 as another IKKK, as judged by two criteria. First, MLK3 expression induced the phosphorylation of kinase-dead versions of IKKα and IKKβ to a comparable extent as did the expression of NIK and MEKK1. Second, immunoprecipitated MLK3 phosphorylated purified GST-IKKα KM and GST-IKKβ KA proteins. All IKKKs identified so far belong to the group of MAPKKKs and are constitutively attached to the IKC (38
). A previous study showed the functional cooperation between NIK and MEKK1 for the activation of IKK activity (55
). Our data indicate that also the MLK3-induced activation of both IKKs is further potentiated by the coexpression of MEKK1 or NIK. In contrast to the MAPKK TAK1, which links TRAF6-derived signals to its downstream target NIK, it seems that MEKK1, NIK, and MLK3 are direct activators of IKKs. Nevertheless, there is a mutual interference between (at least some) of the IKKKs. The MEKK1-induced NF-κB activation was impaired in the presence of dominant negative NIK (54
), and the MLK3-induced activation of IKKs was reduced by kinase-inactive variants of NIK and MEKK1. From a molecular point of view this could be explained by competition of the IKKKs for a common activation site, which would be compatible with the finding that various IKKKs use identical IKK phosphorylation sites. On the other hand, this competition model does not explain the synergistic activation pattern of IKKKs, which suggests that these kinases act in parallel. The simultaneous activation of several IKKKs may help to explain the previous finding that one IKKK alone is not sufficient for full activation of NF-κB. For example, TNF-α-induced NF-κB activation cannot be completely inhibited by dominant negative forms of MEKK1 (37
). It was suggested that MEKK1 has a predilection for IKKβ (54
) and that NIK preferentially phosphorylates IKKα over IKKβ (44
). IKKβ was more efficiently phosphorylated by MLK3 than IKKα. This might be explained by the phosphorylation of IKKα at only one serine, in comparison to IKKβ, which is phosphorylated at two serine residues. Since IKKβ KA repressed MLK3-elicited NF-κB activation more strongly than IKKα KM, it may also be possible that MLK3 preferentially phosphorylates IKKβ. All experiments discussed here used IKKs that were purified by immunoprecipitation, leaving the formal possibility that the phosphorylation might have been caused by a coprecipitating kinase. The tandem leucine zipper of MLK3 is essential for its binding to the IKC in vivo and in vitro. It remains to be seen whether the contact to both IKKs is direct or mediated by binding to intermediate proteins such as NIK, NEMO/IKKγ, or IKAP. MLK3 is also found in association with the scaffold protein JIP, along with other components of a JNK signaling pathway (68
). It was proposed that the simultaneous binding of HPK1, MLK3, MKK7, and JNK to JIP may mediate the coordinate sequential interaction of these kinases (68
), but the ratios between scaffold-attached and free MLK3 are not known. The ectopic expression of MLK3 potently activated NF-κB at low input levels, whereas high doses were ineffective. This may be taken as an indication that only MLK3 that is properly complexed and incorporated into the IKC (and/or the JIP complex) is competent for NF-κB activation. This also might explain the lack of NF-κB induction by ectopic expression of MLK3 described in a recent study (74
), since the authors used rather high amounts of MLK3 expression vector (6 μg).
Since MLK3 does not play an important role in NF-κB activation by TNF-α or IL-1, this study corroborates the concept that different NF-κB-activating stimuli use different MAPKKKs. Along this line, IKKβ−/−
fibroblasts show no TNF-α-induced degradation of IκB-α and activation of NF-κB, but the IL-1-induced IκB phosphorylation remains essentially unaffected (40
). The differential activation of distinct IKKKs would explain earlier findings describing the synergistic activation of NF-κB by the simultaneous addition of distinct NF-κB activators (2
). It will be exciting to learn whether the cell contains distinct sets of differentially composed IKCs, or whether the distinct signaling cascades deliver their signals to uniformly built IKCs, thereby differentially affecting IKKKs.
Earlier studies revealed the activation of NF-κB by Rac/Cdc42-derived signals (58
). Therefore, the identification of MLK3 as an activator of NF-κB uncovers one of the components of the signaling pathway between Rac and NF-κB. Activation of Rac by T-cell costimulation involves Vav family proteins, which act as a GTP/GDP exchange factor for the Rho family of GTPases (5
). The analysis of Vav1−/−
mice revealed that Vav1 is dispensable for CD28 costimulation (57
) but necessary for the CD3/CD28-induced activation of NF-κB (9
). Since MLK3 is widely expressed in a variety of tissues, it is reasonable to assume that MLK3 can be activated by further Rac/Cdc42-dependent stimuli (53
), which await their identification in future studies.