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. Cdkn1b expression is regulated by Mekk1-dependent signaling in differentiated Th17 cells. Mekk1 is one of the 19 Ste11-like Map3ks, and Mekk1 signaling regulates iNKT cell proliferative expansion in response to glycolipid antigens and T cell homeostasis in the liver. Tak1 (encoded by Map3k7), a related Map3k to Mekk1, similarly regulates the proliferative expansion and homeostasis of T cells in the liver, and this illustrates the importance of multiple Map3ks for mammalian Mapk signaling.

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. Lck(Cre/+)Map3k1(f/f) mice have enhanced liver damage in response to glycolipid antigen. Mekk1 regulates Jnk activation in iNKT cells and binds and transfers Lys63-linked poly-ubiquitin onto Carma1. Map3k1 is critical for the regulation of p27(Kip1) (encoded by Cdkn1b).

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-ß)-activated kinase 1 binding protein 1 (Tab1) as a PHD substrate. The Map3k1 PHD transfers Lys63-linked poly-ubiquitin onto Tab1 to activate MAPKs.

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-ß, EGF and microtubule disruption signalling, but does not affect MAPK responses to hyperosmotic stress. Protein microarray profiling identified the adaptor TAB1 as a PHD substrate, and TGF-ß- or EGF-stimulated Map3k1(m) (PHD) ES cells exhibit defective non-canonical ubiquitination of MEKK1 and TAB1. The MEKK1 PHD binds and mediates the transfer of Lys63-linked poly-Ub, using the conjugating enzyme UBE2N, onto TAB1 to regulate TAK1 and MAPK activation by TGF-ß and EGF. Both the MEKK1 PHD and TAB1 are critical for ES-cell differentiation and tumourigenesis. Map3k1(m) (PHD) (/+) mice exhibit aberrant cardiac tissue, B-cell development, testis and T-cell signalling.

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. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival.

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. In contrast, ΔNp73 shows a survival function in mature cortical neurons as selective ΔNp73 null mice have reduced cortical thickness. Recent evidence has also suggested that p73 isoforms are deregulated in neurodegenerative pathologies such as Alzheimer's disease, with abnormal tau phosphorylation. Thus, in addition to its increasingly accepted contribution to tumorigenesis, the p73 subfamily also plays a role in neuronal development and neurodegeneration.

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-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

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. E3 ligase Itch is recruited to activated MEKK1, but not MEKK2, and this novel scaffolding interaction is dependent on MEKK1 Thr(1381) phosphorylation within the kinase domain and an intact MEKK1 RING finger motif. MEKK1 phosphorylation on Thr(1381) is observed during Th2 differentiation, but not under Th1 differentiation. Both Itch and the MEKK1 kinase domain are important for Il4 and Il6 cytokine gene expression under Th2 conditions.

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. Recent work, however, illustrates that protein ubiquitination cascades play a critical initiating role in TNFR signaling and account for spatial and temporal separation of IKK and MAPK signaling cascades and thereby determine biological specificity and outcome. Cellular inhibitors of apoptosis (cIAPs) 1 and 2 are ubiquitin (Ub) ligases (E3s) that mediate canonical Lys48-linked ubiquitination of TNFR-associated factor 3 (TRAF3), marking it for subsequent degradation by the proteasome. TRAF3 degradation releases the brake on TRAF2/6:MAP3K signaling complexes responsible for MAPK activation, leading to their translocation from the cytoplasmic segment of the receptor to the cytosol where they initiate MAPK phosphorylation and activation. By contrast, IKK activation proceeds considerably faster than MAPK activation, takes place at the receptor, and is independent of cIAP1/2 activity and TRAF3 degradation. This arrangement may be important for ensuring the proper delivery of NF-kappaB-dependent survival signals and conversion of JNK-promoted death signals to proliferative ones.

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. However, these kinases were not activated unless the multicomponent signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This two-stage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.

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. MEKK1 was recruited to CD40 and adaptor molecule TRAF2 after CD40 ligation, and Map3k1(deltaKD) B cells were hypoproliferative after CD40 stimulation. Our data emphasize that MEKK1 is an essential component of signaling cascades needed for thymus-dependent antigen-induced B cell proliferation and antibody production.

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. Mutants, whether monomers or dimers, have specific protein kinase activities under fixed assay conditions that are roughly equivalent to wild-type ERK2. The ratio of dimers to monomers is increased as the salt concentration increases, consistent with a strong hydrophobic contribution to the energy of dimer formation. ERK2 dimerization also requires divalent cations. Sedimentation analysis indicates that the related c-Jun N-terminal kinase SAPKalphaI/JNK2 also forms dimers, but dimerization displays no dependence on phosphorylation; the unphosphorylated and phosphorylated forms of the kinase behave similarly, with low micromolar dimer dissociation constants.

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. Phosphorylation of these sites is necessary and sufficient for disrupting an inhibitory interaction between the WW domain of Itch and its catalytic HECT (Homologous to E6-AP C Terminus) domain and induces a conformational change that greatly enhances the catalytic activity of Itch, a HECT E3 ligase found to be directly activated upon its phosphorylation.

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. The JNKs, which are encoded by three different Jnk loci, are now known to be regulated by many other stimuli, from pro-inflammatory cytokines to obesity, in addition to UV-irradiation. Targeted disruption of the Jnk loci in mice has proved to be a critical tool in better understanding their physiological functions. Such studies revealed that the JNKs play important roles in numerous cellular processes, including: programmed cell death, T cell differentiation, negative regulation of insulin signaling, control of fat deposition, and epithelial sheet migration. Importantly, the JNKs have become prime targets for drug development in several important clinical areas, including: inflammation, diabetes, and cancer.

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. Activation of the Jun amino-terminal kinase (JNK) mitogen-activated protein kinase cascade after T cell stimulation accelerated degradation of c-Jun and JunB through phosphorylation-dependent activation of the E3 ligase Itch. This pathway modulates cytokine production by effector T cells.

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. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1.

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). Serine 67 is constitutively phosphorylated in resting 293 cells, but is dephosphorylated following exposure to stress stimuli such as anisomycin and UV irradiation. Phosphorylation of this site inhibits binding of JNK/SAPK to MEKK1. Thus, we propose a mechanism by which the MEKK1-dependent JNK/SAPK pathway is negatively regulated by PAK through phosphorylation of serine 67.

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. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.