Role of mitogen-activated protein kinase kinase 4 in cancer.

Mitogen-activated protein (MAP) kinase kinase 4 (MKK4) is a component of stress activated MAP kinase signaling modules. It directly phosphorylates and activates the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stress, pro-inflammatory cytokines and developmental cues. MKK4 is ubiquitously expressed and the targeted deletion of the Mkk4 gene in mice results in early embryonic lethality. Further studies in mice have indicated a role for MKK4 in liver formation, the immune system and cardiac hypertrophy. In humans, it is reported that loss of function mutations in the MKK4 gene are found in approximately 5% of tumors from a variety of tissues, suggesting it may have a tumor suppression function. Furthermore, MKK4 has been identified as a suppressor of metastasis of prostate and ovarian cancers. However, the role of MKK4 in cancer development appears complex as other studies support a pro-oncogenic role for MKK4 and JNK. Here we review the biochemical and functional properties of MKK4 and discuss the likely mechanisms by which it may regulate the steps leading to the formation of cancers.

c-Jun is a JNK-independent coactivator of the PU.1 transcription factor.

The ETS domain transcription factor PU.1 is necessary for the development of monocytes and regulates, in particular, the expression of the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor, which is critical for monocytic cell survival, proliferation, and differentiation. The bZIP transcription factor c-Jun, which is part of the AP-1 transcription factor complex, is also important for monocytic differentiation, but the monocyte-specific M-CSF receptor promoter has no AP-1 consensus binding sites. We asked the question of whether c-Jun could promote the induction of the M-CSF receptor by collaborating with PU.1. We demonstrate that c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter as well as a minimal thymidine kinase promoter containing only PU.1 DNA binding sites. c-Jun does not directly bind to the M-CSF receptor promoter but associates via its basic domain with the ETS domain of PU.1. Consistent with our observation that AP-1 binding does not contribute to c-Jun coactivation is the observation that the activation of PU.1 by c-Jun is blocked by overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter the ability of c-Jun to enhance PU.1 transactivation. Activated Ras enhances the transcriptional activity of PU.1 by up-regulating c-Jun expression without changing the phosphorylation pattern of PU.1. The activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking the basic domain. The expression of this mutant form of c-Jun also completely blocks 12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor promoter activity during monocytic differentiation. We propose therefore that c-Jun acts as a c-Jun NH2-terminal kinase-independent coactivator of PU.1, resulting in M-CSF receptor expression and development of the monocytic lineage.

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2.

Precursor CD4+ T cells develop into effector Th1 and Th2 cells that play a central role in the immune response. We show that the JNK MAP kinase pathway is induced in Th1 but not in Th2 effector cells upon antigen stimulation. Further, the differentiation of precursor CD4+ T cells into effector Th1 but not Th2 cells is impaired in JNK2-deficient mice. The inability of IL-12 to differentiate JNK2-deficient CD4+ T cells fully into effector Th1 cells is caused by a defect in IFNgamma production during the early stages of differentiation. The addition of exogenous IFNgamma during differentiation restores IL-12-mediated Th1 polarization in the JNK2-deficient mice. The JNK MAP kinase signaling pathway, therefore, plays an important role in the balance of Th1 and Th2 immune responses.

A mammalian scaffold complex that selectively mediates MAP kinase activation.

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by the exposure of cells to multiple forms of stress. A putative scaffold protein was identified that interacts with multiple components of the JNK signaling pathway, including the mixed-lineage group of MAP kinase kinase kinases (MLK), the MAP kinase kinase MKK7, and the MAP kinase JNK. This scaffold protein selectively enhanced JNK activation by the MLK signaling pathway. These data establish that a mammalian scaffold protein can mediate activation of a MAP kinase signaling pathway.

Differential targeting of MAP kinases to the ETS-domain transcription factor Elk-1.

The activation of MAP kinase (MAPK) signal transduction pathways results in the phosphorylation of transcription factors by the terminal kinases in these cascades. Different pathways are activated by mitogenic and stress stimuli, which lead to the activation of distinct groups of target proteins. The ETS-domain transcription factor Elk-1 is a substrate for three distinct classes of MAPKs. Elk-1 contains a targeting domain, the D-domain, which is distinct from the phosphoacceptor motifs and is required for efficient phosphorylation and activation by the ERK MAPKs. In this study, we demonstrate that members of the JNK subfamily of MAPKs are also targeted to Elk-1 by this domain. Targeting via this domain is essential for the efficient and rapid phosphorylation and activation of Elk-1 both in vitro and in vivo. The ERK and JNK MAPKs use overlapping yet distinct determinants in the D-domain for targeting to Elk-1. In contrast, members of the p38 subfamily of MAPKs are not targeted to Elk-1 via this domain. Our data therefore demonstrate that different classes of MAPKs exhibit differential requirements for targeting to Elk-1.

Mechanism of sodium arsenite-mediated induction of heme oxygenase-1 in hepatoma cells. Role of mitogen-activated protein kinases.

Heme oxygenase-1 is an inducible enzyme that catalyzes heme degradation and has been proposed to play a role in protecting cells against oxidative stress-related injury. We investigated the induction of heme oxygenase-1 by the tumor promoter arsenite in a chicken hepatoma cell line, LMH. We identified a heme oxygenase-1 promoter-driven luciferase reporter construct that was highly and reproducibly expressed in response to sodium arsenite treatment. This construct was used to investigate the role of mitogen-activated protein (MAP) kinases in arsenite-mediated heme oxygenase-1 gene expression. In LMH cells, sodium arsenite, cadmium, and heat shock, but not heme, induced activity of the MAP kinases extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. To examine whether these MAP kinases were involved in mediating heme oxygenase-1 gene expression, we utilized constitutively activated and dominant negative components of the ERK, JNK, and p38 MAP kinase signaling pathways. Involvement of an AP-1 site in arsenite induction of heme oxygenase-1 gene expression was studied. We conclude that the MAP kinases ERK and p38 are involved in the induction of heme oxygenase-1, and that at least one AP-1 element (located -1576 base pairs upstream of the transcription start site) is involved in this response.

The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif.

The phosphorylation of transcription factors by mitogen-activated protein kinases (MAP) is a pivotal event in the cellular response to the activation of MAP kinase signal transduction pathways. Mitogenic and stress stimuli activate different pathways and lead to the activation of distinct groups of target proteins. Elk-1 is targeted by three distinct MAP kinase pathways. In this study, we demonstrate that the MAP kinase ERK2 is targeted to Elk-1 by a domain which is distinct from, and located N-terminally to, its phosphoacceptor motifs. Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full and rapid activation in vivo. Specific residues involved in ERK targeting have been identified. Our data indicate that the targeting of different classes of MAP kinases to their nuclear substrates may be a common mechanism to increase the specificity and efficiency of this signal transduction pathway.

Phosphorylation of the oncogenic transcription factor interferon regulatory factor 2 (IRF2) in vitro and in vivo.

IRF2 is a transcription factor, possessing oncogenic potential, responsible for both the repression of growth-inhibiting genes (interferon) and the activation of cell cycle-regulated genes (histone H4). Surprisingly little is known about the post-translational modification of this factor. In this study, we analyze the phosphorylation of IRF2 both in vivo and in vitro. Immunoprecipitation of HA-tagged IRF2 expressed in 32P-phosphate labelled COS-7 cells demonstrates that IRF2 is phosphorylated in vivo. Amino acid sequence analysis reveals that several potential phosphorylation sites exist for a variety of serine/threonine protein kinases, including those of the mitogen activated protein (MAP) kinase family. Using a battery of these protein kinases we show that recombinant IRF2 is a substrate for protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CK2) in vitro. However, other serine/threonine protein kinases, including the MAP kinases JNK1, p38, and ERK2, do not phosphorylate IRF2. Two-dimensional phosphopeptide mapping of the sites phosphorylated by PKA, PKC, and CKII in vitro demonstrates that these enzymes are capable of phosphorylating IRF2 at multiple distinct sites. Phosphoaminoacid analysis of HA-tagged IRF2 immunoprecipitated from an asynchronous population of proliferating, metabolically phosphate-labelled cells indicates that this protein is phosphorylated exclusively upon serine residues in vivo. These results suggest that the oncogenic protein IRF2 may be regulated via multiple pathways during cellular growth.

Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors.

The transcription factors Elk-1 and SAP-1 bind together with serum response factor to the serum response element present in the c-fos promoter and mediate increased gene expression. The ERK, JNK, and p38 groups of mitogen-activated protein (MAP) kinases phosphorylate and activate Elk-1 in response to a variety of extracellular stimuli. In contrast, SAP-1 is activated by ERK and p38 MAP kinases but not by JNK. The proinflammatory cytokine interleukin-1 (IL-1) activates JNK and p38 MAP kinases and induces the transcriptional activity of Elk-1 and SAP-1. These effects of IL-1 appear to be mediated by Rho family GTPases. To examine the relative roles of the JNK and p38 MAP kinase pathways, we examined the effects of IL-1 on CHO and NIH 3T3 cells. Studies of NIH 3T3 cells demonstrated that both the JNK and p38 MAP kinases are required for IL-1-stimulated Elk-1 transcriptional activity, while only p38 MAP kinase contributes to IL-1-induced activation of SAP-1. In contrast, studies of CHO cells demonstrated that JNK (but not the p38 MAP kinase) is required for IL-1-stimulated Elk-1-dependent gene expression and that neither JNK nor p38 MAP kinase is required for IL-1 signaling to SAP-1. We conclude that (i) distinct MAP kinase signal transduction pathways mediate IL-1 signaling to ternary complex transcription factors (TCFs) in different cell types and (ii) individual TCFs show different responses to the JNK and p38 signaling pathways. The differential utilization of TCF proteins and MAP kinase signaling pathways represents a potential mechanism for the determination of cell-type-specific responses to extracellular stimuli.

Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene.

Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is an important component in the pathogenesis of glutamate neurotoxicity.

Determinants of DNA-binding specificity of ETS-domain transcription factors.

Several mechanisms are employed by members of transcription factor families to achieve sequence-specific DNA recognition. In this study, we have investigated how members of the ETS-domain transcription factor family achieve such specificity. We have used the ternary complex factor (TCF) subfamily as an example. ERK2 mitogen-activated protein kinase stimulates serum response factor-dependent and autonomous DNA binding by the TCFs Elk-1 and SAP-la. Phosphorylated Elk-1 and SAP-la exhibit specificities of DNA binding similar to those of their isolated ETS domains. The ETS domains of Elk-1 and SAP-la and SAP-2 exhibit related but distinct DNA-binding specificities. A single residue, D-69 (Elk-1) or V-68 (SAP-1), has been identified as the critical determinant for the differential binding specificities of Elk-1 and SAP-1a, and an additional residue, D-38 (Elk-1) or Q-37 (SAP-1), further modulates their DNA binding. Creation of mutations D38Q and D69V is sufficient to confer SAP-la DNA-binding specificity upon Elk-1 and thereby allow it to bind to a greater spectrum of sites. Molecular modelling indicates that these two residues (D-38 and D-69) are located away from the DNA-binding interface of Elk-1. Our data suggest a mechanism in which these residues modulate DNA binding by influencing the interaction of other residues with DNA.

Selective interaction of JNK protein kinase isoforms with transcription factors.

The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.

Integration of MAP kinase signal transduction pathways at the serum response element.

The ternary complex factor (TCF) subfamily of ETS-domain transcription factors bind with serum response factor (SRF) to the serum response element (SRE) and mediate increased gene expression. The TCF protein Elk-1 is phosphorylated by the JNK and ERK groups of mitogen-activated protein (MAP) kinases causing increased DNA binding, ternary complex formation, and transcriptional activation. Activated SRE-dependent gene expression is induced by JNK in cells treated with interleukin-1 and by ERK after treatment with phorbol ester. The Elk-1 transcription factor therefore integrates MAP kinase signaling pathways in vivo to coordinate biological responses to different extracellular stimuli.