The HEP protein is homologous to the Jun kinase kinase (JNKK) group of mitogen-activated protein kinase kinases (MAPKKs). Highest homology in the kinase domain is to human MKK4 and Xenopus XMEK2 with a 56% identity to both. Homology to the fly protein DSOR1 is 40% (Glise, 1995).

At least three mitogen-activated protein kinase (MAPK) cascades have been identified in mammals, each consisting of a well-defined three-kinase module composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). These cascades play key roles in relaying various physiological, environmental, or pathological signals from the environment to the transcriptional machinery in the nucleus. One of these MAPKs, c-Jun N-terminal kinase (JNK), stimulates the transcriptional activity of c-Jun in response to growth factors, proinflammatory cytokines, and certain environmental stresses, such as short wavelength UV light or osmotic shock. The JNKs are directly activated by the MAPKK JNKK1/SEK1/MKK4. However, inactivation of the gene encoding this MAPKK by homologous recombination suggests the existence of at least one more JNK-activating kinase. Recently, the JNK cascade was found to be structurally and functionally conserved in Drosophila, where DJNK is activated by the MAPKK DJNKK (Hemipterous). By a database search, an expressed sequence tag (EST) was identified encoding a portion of human MAPKK that is highly related to DJNKK (hep). This EST was used to isolate a full-length cDNA clone encoding a human JNKK2. JNKK2 is a highly specific JNK kinase. Unlike JNKK1, it does not activate the related MAPK, p38. Although the regulation of both JNKK1 and JNKK2 activities could be very similar, the two kinases may play somewhat different regulatory roles in a cell-type-dependent manner (Wu, 1997).

Exposure of mammalian cells to stressful stimuli results in activation of the c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinases (SAPKs), a family of protein kinases related to mitogen-activated protein (MAP) kinase. JNK/SAPKs are activated by specific MAP kinase kinases (MKKs), one of which, MKK4/SEK1, has been characterized extensively. In Drosophila, the JNK/SAPK Basket (Bsk) and the MKK Hemipterous (Hep), are important for embryonic development. Loss of function of either gene inhibits dorsal closure, a morphogenetic movement in which the edges of the embryonic ectoderm move together over the amnioserosa. There is evidence that the Rho GTPases Rac and Cdc42 are also required for dorsal closure, suggesting that Rac or Cdc42 may regulate Hep and Bsk. MKK7, a murine homolog of Hep, has been identified. MKK7 functionally rescues hep mutant flies. In fibroblasts, MKK7 is activated by stress and by the GTPase Rac1. MKK7 directly phosphorylates and activates JNK/SAPK. Thus, MKK7 is a homolog of hep and functions in a conserved signaling pathway involving JNK/SAPK and the GTPase Rac1 (Holland, 1997).

The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. sek2/mkk7 has greater sequence homology to hemipterous and SEK2 is highly expressed in epithelial tissues. The sek1 gene as been disrupted in mice using homologous recombination. Sek1(-/-) embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Hematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/-) embryos. However, hepatogenesis and liver formation are severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/-) embryos have greatly reduced numbers of parenchymal hepatocytes. Although formation of the primordial liver from the visceral endoderm appears normal, sek1(-/-) liver cells undergo massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes. While the SEK1->SAPK/JNK->c-Jun signaling cascade is a common pathway required for the induction of apoptosis in response to many types of stress, here it is shown that SEK1 provides a survival signal for hepatocytes (Nishina, 1999).

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. The molecular cloning of the gene that encodes MKK7 is reported and it is demonstrated that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus is not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli (Tournier, 1999).

MAP kinase (MAPK) cascades are composed of a MAPK, MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Despite the existence of numerous components and ample opportunities for crosstalk, most MAPKs are specifically and distinctly activated. The basis for the specific activation of the JNK subgroup of MAPKs has been studied. The specificity of JNK activation is determined by the MAPKK JNKK1, which interacts with the MAPKKK MEKK1 and JNK through its amino-terminal extension. Inactive JNKK1 mutants can disrupt JNK activation by MEKK1 or tumor necrosis factor (TNF) in intact cells only if the mutants contain an intact amino-terminal extension. Mutations in this region interfere with the ability of JNKK1 to respond to TNF but do not affect its activation by physical stressors. As JNK and MEKK1 compete for binding to JNKK1 and activation of JNKK1 prevents its binding to MEKK1, activation of this module is likely to occur through sequential MEKK1:JNKK1 and JNKK1:JNK interactions. These results underscore a role for the amino-terminal extension of MAPKKs in determination of response specificity (Xia, 1998).

Stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, is thought to play a key role in a variety of cellular responses. To date, SEK1/MKK4, one of the MAP kinase kinase (MAPKK) family of molecules, is the only SAPK/JNK kinase that has been cloned. A novel member of the mammalian MAPKKs, designated MKK7, has now been cloned, identified and characterized. MKK7 is most similar to the Drosophila mediator of morphogenesis, hemipterous (hep). Immunochemical studies have identified MKK7 as one of the major SAPK/JNK-activating kinases in osmotically shocked cells. While SEK1/MKK4 can activate both the SAPK/JNK and p38 subgroups of the MAPK superfamily, MKK7 is specific for the SAPK/JNK subgroup. MKK7 is activated strongly by tumour necrosis factor alpha (TNFalpha) as well as by environmental stresses, whereas SEK1/MKK4 is not activated by TNFalpha. Column fractionation studies have shown that MKK7 is a major activator for SAPK/JNK in the TNFalpha-stimulated pathway. It is found that overexpression of MKK7 enhances transcription from an AP-1-dependent reporter construct. Thus, MKK7 is an evolutionarily conserved MAPKK isoform, specific for SAPK/JNK, involved in AP-1-dependent transcription and may be a crucial mediator of TNFalpha signaling (Moriguchi, 1997).

Mitogen-activated protein kinase kinase 7 (MKK7), a novel human activator of c-Jun N-terminal kinase (JNK) and widely expressed in humans and mice, is a 47-kDa protein (419 amino acids). The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and is more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylates and activates JNK1 but fails to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 is activated either by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of either CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 is also activated when cells are exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-alpha. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activates MKK7, suggesting that MKK7 is involved in many physiological pathways (Foltz, 1998).

A pleiotropic cytokine, tumor necrosis factor-alpha (TNF alpha), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. The specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages has been investigated in response to stimulation with TNF alpha. Exposure of macrophages to TNF alpha stimulates a preferential increase in catalytic activity of the p46 JNK/SAPK isoform, as compared with the p54 JNK/SAPK isoform. To investigate the level of regulation of p46 JNK/SAPK activation, an examination was made of the ability of MKK4/SEK1/JNKK (an upstream regulator of JNK/SAPKs) to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 is able to transphosphorylate both isoforms. Both the p46 and p54 JNK/SAPK isoforms are phosphorylated on their TPY motif in response to TNF alpha stimulation. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNF alpha regulates macrophage phenotypic heterogeneity and differentiation (Chan, 1997).

The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA does not exhibit this activity, and none of the proteins activated the ERK subgroup of MAPKs. JNK, but not ERK, activation iss also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation (Minden, 1995).

The mammalian p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, expression vectors were constructed for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells cause a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrate that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway (Raingeaud, 1995).

The c-Jun NH2-terminal kinase (JNK) has been implicated in both cell death and survival responses to different stimuli. This study reexamines the function of JNK in tumor necrosis factor (TNF)-stimulated cell death using fibroblasts isolated from wild-type, Mkk4-/- Mkk7-/-, and Jnk1-/- Jnk2-/- mice. JNK can act to suppress TNF-stimulated apoptosis. However, JNK can also potentiate TNF-stimulated necrosis by increasing the production of reactive oxygen species (ROS). Together, these data indicate that JNK can shift the balance of TNF-stimulated cell death from apoptosis to necrosis. Increased necrosis may represent a contributing factor in stress-induced inflammatory responses mediated by JNK (Ventura, 2004).

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