Mixed lineage kinase LZK forms a functional signaling complex with JIP-1, a scaffold protein of the c-Jun NH(2)-terminal kinase pathway.

Leucine zipper-bearing kinase (LZK) is a novel member of the mixed lineage kinase (MLK) protein family, the cDNA of which was first cloned from a human brain cDNA library [Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J.-P., and Kawasaki, T. (1997) J. Biol. Chem. 272, 28622-28629]. Several MLK family proteins have been proposed to function as MAP kinase kinase kinases in the c-Jun NH(2) terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway. In the present study, we demonstrated that, like other MLKs, LZK activated the JNK/SAPK pathway but not the ERK pathway. LZK directly phosphorylated and activated MKK7, one of the two MAPKKs in the JNK/SAPK pathway, to a comparable extent to a constitutive active form of MEKK1 (MEKK1DeltaN), suggesting a biological role of LZK as a MAPKKK in the JNK/SAPK pathway. Recent studies have revealed the essential roles of scaffold proteins in intracellular signaling pathways including MAP kinase pathways. JIP-1, one of the scaffold proteins, has been shown to be associated with MLKs, MKK7, and JNK [Whitmarsh, A.J., Cavanagh, J., Tournier, C., Yasuda, J., and Davis, R.J. (1998) Science 281, 1671-1674], suggesting the presence of a selective signaling pathway including LZK, MKK7, and JNK. Consistent with this hypothesis, we provided evidence that LZK is associated with the C-terminal region of JIP-1 through its kinase catalytic domain. In addition, LZK-induced JNK activation was markedly enhanced when LZK and JNK were co-expressed with JIP-1. These results constituted important clues for understanding the molecular mechanisms regulating the signaling specificities of various JNK activators under different cellular conditions.     

FAF1 suppresses IkappaB kinase (IKK) activation by disrupting the IKK complex assembly

This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IkappaB kinase (IKK) activation, where divergent NF-kappaB-activating stimuli converge. FAF1 interacts with IKKbeta in response to proinflammatory stimuli (such as tumor necrosis factor-alpha, interleukin-1beta, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKbeta with FAF1 affected the IKK heterocomplex (IKKalpha/beta) and homocomplex (IKKalpha/alpha, IKKbeta/beta) formations and attenuated IKKgamma recruitment to IKKbeta. Overexpression of FAF1 reduced the level of IKKbeta activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKbeta. Taken together, FAF1 robustly suppresses NF-kappaB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-kappaB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544-2549). Such redundant suppression would prevent inadvertent activation of the NF-kappaB pathway.     

Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via post-synaptic density protein 95

Kainate receptor glutamate receptor 6 (GluR6) subunit-deficient and c-Jun N-terminal kinase 3 (JNK3)-null mice share similar phenotypes including resistance to kainite-induced epileptic seizures and neuronal toxicity (Yang, D. D., Kuan, C-Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakis, P., and Flavell, R. (1997) Nature 389, 865-869; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). This suggests that JNK activation may be involved in GluR6-mediated excitotoxicity. We provide evidence that post-synaptic density protein (PSD-95) links GluR6 to JNK activation by anchoring mixed lineage kinase (MLK) 2 or MLK3, upstream activators of JNKs, to the receptor complex. Association of MLK2 and MLK3 with PSD-95 in HN33 cells and rat brain preparations is dependent upon the SH3 domain of PSD-95, and expression of GluR6 in HN33 cells activated JNKs and induced neuronal apoptosis. Deletion of the PSD-95-binding site of GluR6 reduced both JNK activation and neuronal toxicity. Co-expression of dominant negative MLK2, MLK3, or mitogen-activated kinase kinase (MKK) 4 and MKK7 also significantly attenuated JNK activation and neuronal toxicity mediated by GluR6, and co-expression of PSD-95 with a deficient Src homology 3 domain also inhibited GluR6-induced JNK activation and neuronal toxicity. Our results suggest that PSD-95 plays a critical role in GluR6-mediated JNK activation and excitotoxicity by anchoring MLK to the receptor complex.     

Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop

Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.     

Signaling pathways to the assembly of an interferon-beta enhanceosome. Chemical genetic studies with a small molecule

Small molecules that modulate specific protein functions are valuable tools for dissecting complex signaling pathways. Here, we identified a small molecule that induces the assembly of the interferon-beta (IFN-beta) enhanceosome by stimulating all the enhancer-binding activator proteins: ATF2/c-JUN, IRF3, and p50/p65 of NF-kappaB. This compound stimulates mitogen-activated protein kinase kinase kinase 1 (MEKK1), which is a member of a family of proteins involved in stress-mediated signaling pathways. Consistent with this, MEKK1 activates IRF3 in addition to ATF2/c-JUN and NF-kappaB for the assembly of the IFN-beta enhanceosome. MEKK1 activates IRF3 through the c-JUN amino-terminal kinase (JNK) pathway but not the p38 and IkappaB kinase (IKK) pathway. Taken together with previous observations, these results implicate that, for the assembly of an IFN-beta enhanceosome, MEKK1 can induce IRF3 and ATF2/c-JUN through the JNK pathway, whereas it can induce NF-kappaB through the IKK pathway. Thus, specific MEKK family proteins may be able to integrate some of multiple signal transduction pathways leading to the specific activation of the IFN-beta enhanceosome.     

Kaposi's sarcoma-associated herpesvirus (KSHV) oncoprotein K13 bypasses TRAFs and directly interacts with the IkappaB kinase complex to selectively activate NF-kappaB without JNK activation

Kaposi's sarcoma herpesvirus oncoprotein vFLIP K13 is a potent activator of NF-kappaB and plays a key role in viral pathogenesis. K13 contains a putative TRAF-interacting motif, which is reportedly required for its interaction with TRAF2. The K13-TRAF2 interaction is believed to be essential for the recruitment of K13 to the I-kappaB kinase (IKK) complex and for K13-induced NF-kappaB and JNK activation. In addition, TRAF3 has been reported to be required for K13-induced NF-kappaB and JNK activation. We have re-examined the role of the TRAFs in K13 signaling and report that mutations in the putative TRAF-interacting motif of K13 have no deleterious effect on its ability to interact with the IKK complex or activation of the NF-kappaB pathway. Furthermore, endogenously expressed TRAF2 and TRAF3 do not interact with K13 and play no role in K13-induced NF-kappaB activation or its interaction with the IKK complex. Finally, K13 does not activate the JNK pathway. Our results support a model in which K13 bypasses the upstream components of the tumor necrosis factor receptor signaling pathway and directly interacts with the IKK complex to selectively activate the NF-kappaB pathway without affecting the JNK pathway. Selective NF-kappaB activation by K13 might represent a novel strategy employed by the virus to promote latency.     

Role of MLK3 in the regulation of mitogen-activated protein kinase signaling cascades

Mixed-lineage protein kinase 3 (MLK3) is a member of the mitogen-activated protein (MAP) kinase kinase kinase group that has been implicated in multiple signaling cascades, including the NF-kappaB pathway and the extracellular signal-regulated kinase, c-Jun NH(2)-terminal kinase (JNK), and p38 MAP kinase pathways. Here, we examined the effect of targeted disruption of the murine Mlk3 gene. Mlk3(-/-) mice were found to be viable and healthy. Primary embryonic fibroblasts prepared from these mice exhibited no major signaling defects. However, we did find that MLK3 deficiency caused a selective reduction in tumor necrosis factor (TNF)-stimulated JNK activation. Together, these data demonstrate that MLK3 contributes to the TNF signaling pathway that activates JNK.     

Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK

The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.     

The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of IkappaB kinase and c-Jun N-terminal kinase

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-kappaB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IkappaB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.     

Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin

We previously reported that expression of polyglutamine-expanded huntingtin induces apoptosis via c-Jun amino-terminal kinase (JNK) activation in HN33 cells (Liu, Y. F. (1998) J. Biol. Chem. 273, 28873-28822). Extending this study, we now demonstrate a role of mixed-lineage kinase 2 (MLK2), a JNK activator, in polyglutamine-expanded huntingtin-mediated neuronal toxicity. We find that normal huntingtin interacts with MLK2, whereas the polyglutamine expansion interferes with this interaction. Similar to the expression of polyglutamine-expanded huntingtin, expression of MLK2 also induces JNK activation and apoptosis in HN33 cells. Co-expression of dominant negative MLK2 significantly attenuates neuronal apoptosis induced by the mutated huntingtin. Furthermore, over-expression of the N terminus of normal huntingtin partially rescues the neuronal toxicity induced by MLK2. Our results suggest that activation of MLK2-mediated signaling cascades may be partially involved in neuronal death induced by polyglutamine-expanded huntingtin.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

Activation of NF-kappa B by bradykinin through a Galpha(q)- and Gbeta gamma-dependent pathway that involves phosphoinositide 3-kinase and Akt

Recent work has suggested a role for the serine/threonine kinase Akt and IkappaB kinases (IKKs) in nuclear factor (NF)-kappaB activation. In this study, the involvement of these components in NF-kappaB activation through a G protein-coupled pathway was examined using transfected HeLa cells that express the B2-type bradykinin (BK) receptor. The function of IKK2, and to a lesser extent, IKK1, was suggested by BK-induced activation of their kinase activities and by the ability of their dominant negative mutants to inhibit BK-induced NF-kappaB activation. BK-induced NF-kappaB activation and IKK2 activity were markedly inhibited by RGS3T, a regulator of G protein signaling that inhibits Galpha(q), and by two Gbetagamma scavengers. Co-expression of Galpha(q) potentiated BK-induced NF-kappaB activation, whereas co-expression of either an activated Galpha(q)(Q209L) or Gbeta(1)gamma(2) induced IKK2 activity and NF-kappaB activation without BK stimulation. BK-induced NF-kappaB activation was partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that PI3K is a downstream effector of Galpha(q) and Gbeta(1)gamma(2) for NF-kappaB activation. Furthermore, BK could activate the PI3K downstream kinase Akt, whereas a catalytically inactive mutant of Akt inhibited BK-induced NF-kappaB activation. Taken together, these findings suggest that BK utilizes a signaling pathway that involves Galpha(q), Gbeta(1)gamma(2), PI3K, Akt, and IKK for NF-kappaB activation.