The PHD motif of Map3k1 activates cytokine-dependent MAPK signaling.

We generated a mutation in the gene encoding mitogen-activated protein kinase kinase kinase 1 (Map3k1) that results in a protein with an inactive plant homeodomain (PHD). Map3k1(mPHD) cells are defective in cytokine-mediated MAPK signaling. Protein array identified transforming growth factor (TGF-β)-activated kinase 1 binding protein 1 (Tab1) as a PHD substrate.

Genetic insights into Map3k-dependent proliferative expansion of T cells.

Mapks are important regulators of T cell proliferative expansion and cell cycle progression. Detailed genetic analysis of unconventional iNKT cells in both Map3k1(ΔKD) and Lck(Cre/+)Map3k1(f/f) mice demonstrated that Mekk1 (encoded by Map3k1) signaling activates Mapks to regulate Cdkn1b (encoding p27(Kip1)) expression and p27(Kip1)-dependent proliferative expansion in response to antigen. Mekk1 signaling and activation of E3 ubiquitin ligase Itch, by a phosphorylation-dependent conformational change, is also an important regulatory mechanism for the control of T helper cell cytokine production.

T-Cell-Specific Deletion of Map3k1 Reveals the Critical Role for Mekk1 and Jnks in Cdkn1b-Dependent Proliferative Expansion.

MAPK signaling is important for T lymphocyte development, homeostasis, and effector responses. To better understand the role of Mekk1 (encoded by Map3k1) in T cells, we conditionally deleted Map3k1 in Lck(Cre/+)Map3k1(f/f) mice, and these display larger iNKT cell populations within the liver, spleen, and bone marrow. Mekk1 signaling controls splenic and liver iNKT cell expansion in response to glycolipid antigen.

The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines.

Unlike the other MAP3Ks, MEKK1 (encoded by Map3k1) contains a PHD motif. To understand the role of this motif, we have created a knockin mutant of mouse Map3k1 (Map3k1(m) (PHD)) with an inactive PHD motif. Map3k1(m) (PHD) ES cells demonstrate that the MEKK1 PHD controls p38 and JNK activation during TGF-β, EGF and microtubule disruption signalling, but does not affect MAPK responses to hyperosmotic stress.

MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival.

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1-c-Jun NH2-terminal kinase (JNK) pathway.

p73: a multifunctional protein in neurobiology.

p73, a transcription factor of the p53 family, plays a key role in many biological processes including neuronal development. Indeed, mice deficient for both TAp73 and ΔNp73 isoforms display neuronal pathologies, including hydrocephalus and hippocampal dysgenesis, with defects in the CA1-CA3 pyramidal cell layers and the dentate gyrus. TAp73 expression increases in parallel with neuronal differentiation and its ectopic expression induces neurite outgrowth and expression of neuronal markers in neuroblastoma cell lines and neural stem cells, suggesting that it has a pro-differentiation role.

Apoptosis induced by cytoskeletal disruption requires distinct domains of MEKK1.

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1.

MEKK1 binds HECT E3 ligase Itch by its amino-terminal RING motif to regulate Th2 cytokine gene expression.

MEKK1-dependent signaling regulates HECT E3 ligase Itch, resulting in elevated catalytic activity. After TCR costimulation, MEKK1 predominantly induces JNK1 activation, whereas the related kinase MEKK2 regulates ERK5 activation. MEKK1 becomes phosphorylated on multiple sites and polyubiquitinated following TCR costimulation.

TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes.

Nearly two decades after the initial cloning and identification of the founding father of the tumor necrosis factor receptor (TNFR) family, much has been learned about the mechanisms by which these receptors signal to critical transcription factors and other targets that regulate gene expression and cellular physiology. Mitogen-activated protein kinases (MAPKs) and inhibitor of nuclear factor (NF)-kappaB (I kappaB) kinases (IKKs) were identified early on as the upstream kinases responsible for activation of activator-protein 1 (AP-1) and NF-kappaB, respectively, and later on for their ability to control life-or-death decisions in TNF-stimulated cells. Both of these critical pathways are regulated at the level of MAPK kinase kinases (MAP3Ks), after which point they diverge.

Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex.

Cytokine signaling is thought to require assembly of multicomponent signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin-conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IkappaB kinase regulatory subunit IKKgamma (also called NEMO), and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKgamma were required for complex assembly and activation of MEKK1 and MAPK cascades.

Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production.

Mice lacking activity of the kinase MEKK1 ('Map3k1(deltaKD)' mice) have defective activation of the kinase Jnk and increased production of T helper type 2 cytokines after T cell receptor ligation. Here we show that Map3k1(deltaKD) mice had defective germinal center formation and diminished production of antibodies recognizing thymus-dependent antigens. Those defects were B cell intrinsic, as MEKK1 was necessary for CD40-mediated activation of the kinases Jnk and p38 and transcription factor c-Jun, as well as for expression of cyclin D2 and activation-induced deaminase.

Characterization of mitogen-activated protein kinase (MAPK) dimers.

Phosphorylated ERK2 has an increased capacity to form homodimers relative to unphosphorylated ERK2. We have characterized the nature of the ERK2 dimer and have mutated residues in the crystal dimer interface to examine the impact of dimerization on ERK2 activity. Analysis of the mutants by gel filtration indicates that at least five residues must be mutated simultaneously to produce an ERK2 mutant that is predominantly monomeric.

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change.

The E3 ubiquitin (Ub) ligase Itch is a critical regulator of T helper 2 (Th2) cytokine production through its ability to induce Ub-dependent JunB degradation. After T cell receptor engagement, Itch undergoes JNK1-mediated phosphorylation that greatly enhances its enzymatic activity. To investigate how phosphorylation activates an E3 Ub ligase we have identified the JNK1 phosphorylation sites within Itch as S199, S232, and T222, which are located within a Pro-rich region.

From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance.

The c-Jun N-terminal kinases (JNKs) were originally identified by their ability to phosphorylate c-Jun in response to UV-irradiation, but now are recognized as critical regulators of various aspects of mammalian physiology, including: cell proliferation, cell survival, cell death, DNA repair and metabolism. JNK-mediated phosphorylation enhances the ability of c-Jun, a component of the AP-1 transcription factor, to activate transcription, in response to a plethora of extracellular stimuli. The JNK activation leads to induction of AP-1-dependent target genes involved in cell proliferation, cell death, inflammation, and DNA repair.

Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch.

The turnover of Jun proteins, like that of other transcription factors, is regulated through ubiquitin-dependent proteolysis. Usually, such processes are regulated by extracellular stimuli through phosphorylation of the target protein, which allows recognition by F box-containing E3 ubiquitin ligases. In the case of c-Jun and JunB, we found that extracellular stimuli also modulate protein turnover by regulating the activity of an E3 ligase by means of its phosphorylation.

RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity.

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades.

Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation.

We sought to characterize the role of upstream kinases in the regulation of the MAP3 kinase MEKK1 and the potential impact on signaling to MAP kinase cascades. We find that the MAP4 kinase PAK1 phosphorylates the amino terminus of MEKK1 on serine 67. We show that serine 67 lies in a D domain, which binds to the c-Jun-NH(2)-terminal kinase/stress-activated protein kinases (JNK/SAPK).

p115 Rho GTPase activating protein interacts with MEKK1.

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function.