Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma.

We have examined the structure and expression of the products associated with the t(2;13)(q35;q14) translocation associated with alveolar rhabdomyosarcoma. The chromosome 13 gene (FKHR) is identified as a member of the fork head domain family of transcription factors characterized by a conserved DNA binding motif. Polymerase chain reaction analysis demonstrates that a 5'PAX3-3' FKHR chimaeric transcript is expressed in all eight alveolar rhabdomyosarcomas investigated. Immunoprecipitation experiments detect the predicted fusion protein. These findings indicate that the t(2;13) generates a potentially tumorigenic fusion transcription factor consisting of intact PAX3 DNA binding domains, a truncated fork head DNA binding domain and C-terminal FKHR regions.

An asymmetric shock wave in the 2006 outburst of the recurrent nova RS Ophiuchi.

Nova outbursts take place in binary star systems comprising a white dwarf and either a low-mass Sun-like star or, as in the case of the recurrent nova RS Ophiuchi, a red giant. Although the cause of these outbursts is known to be thermonuclear explosion of matter transferred from the companion onto the surface of the white dwarf, models of the previous (1985) outburst of RS Ophiuchi failed to adequately fit the X-ray evolution and there was controversy over a single-epoch high-resolution radio image, which suggested that the remnant was bipolar rather than spherical as modelled. Here we report the detection of spatially resolved structure in RS Ophiuchi from two weeks after its 12 February 2006 outburst. We track an expanding shock wave as it sweeps through the red giant wind, producing a remnant similar to that of a type II supernova but evolving over months rather than millennia. As in supernova remnants, the radio emission is non-thermal (synchrotron emission), but asymmetries and multiple emission components clearly demonstrate that contrary to the assumptions of spherical symmetry in models of the 1985 explosion, the ejection is jet-like, collimated by the central binary whose orientation on the sky can be determined from these observations.

Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development.

The c-Jun amino-terminal kinase (JNK) group of MAP kinases has been identified in mammals and insects. JNK is activated by exposure of cells to cytokines or environmental stress, indicating that this signaling pathway may contribute to inflammatory responses. Genetic and biochemical studies demonstrate that this signaling pathway also regulates cellular proliferation, apoptosis, and tissue morphogenesis. A functional role for JNK is therefore established in both the cellular response to stress and in many normal physiological processes.

Spinal muscular atrophy disrupts the interaction of ZPR1 with the SMN protein.

The survival motor neurons (smn) gene in mice is essential for embryonic viability. In humans, mutation of the telomeric copy of the SMN1 gene causes spinal muscular atrophy, an autosomal recessive disease. Here we report that the SMN protein interacts with the zinc-finger protein ZPR1 and that these proteins colocalize in small subnuclear structures, including gems and Cajal bodies. SMN and ZPR1 redistribute from the cytoplasm to the nucleus in response to serum. This process is disrupted in cells from patients with Werdnig-Hoffman syndrome (spinal muscular atrophy type I) that have SMN1 mutations. Similarly, decreased ZPR1 expression prevents SMN localization to nuclear bodies. Our data show that ZPR1 is required for the localization of SMN in nuclear bodies.

Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.

Regulation of c-Jun NH(2)-terminal kinase (Jnk) gene expression during T cell activation.

The c-Jun NH(2)-terminal kinases (JNKs) are a group of mitogen-activated protein (MAP) kinases that participate in signal transduction events mediating specific cellular functions. Activation of JNK is regulated by phosphorylation in response to cellular stress and inflammatory cytokines. Here, we demonstrate that JNK is regulated by a second, novel mechanism. Induction of Jnk gene expression is required in specific tissues before activation of this signaling pathway. The in vivo and in vitro ligation of the T cell receptor (TCR) leads to induction of JNK gene and protein expression. TCR signals are sufficient to induce JNK expression, whereas JNK phosphorylation also requires CD28-mediated costimulatory signals. Therefore, both expression and activation contribute to the regulation of the JNK pathway to ensure proper control during the course of an immune response.

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes.

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.

Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo.

The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is strictly regulated during the differentiation of CD4(-)CD8(-) thymocytes. Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25(+)CD44(-) stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice. Inactivation of p38 MAP kinase is required for further differentiation of these cells into CD4(+)CD8(+) thymocytes. The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation. Therefore, the p38 MAP kinase pathway is a critical regulatory element of differentiation and proliferation during the early stages of in vivo thymocyte development.

Role of MKK3-p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice.

AIMS/HYPOTHESIS: Obesity and diabetes are associated with increased intracellular p38 mitogen-activated protein kinase (MAPK) signalling, which may promote tissue inflammation and injury. Activation of p38 MAPK can be induced by either of the immediate upstream kinases, MAP kinase kinase (MKK)3 or MKK6, and recent evidence suggests that MKK3 has non-redundant roles in the pathology attributed to p38 MAPK activation. Therefore, this study examined whether MKK3 signalling influences the development of obesity, type 2 diabetes and diabetic nephropathy. METHODS: Wild-type and Mkk3 (also known as Map2k3) gene-deficient db/db mice were assessed for the development of obesity, type 2 diabetes and renal injury from 8 to 32 weeks of age. RESULTS: Mkk3 (+/+) db/db and Mkk3 (-/-) db/db mice developed comparable obesity and were similar in terms of incidence and severity of type 2 diabetes. At 32 weeks, diabetic Mkk3 (+/+) db/db mice had increased kidney levels of phospho-p38 and MKK3 protein. In comparison, kidney levels of phospho-p38 in diabetic Mkk3 ( -/- ) db/db mice remained normal, despite a fourfold compensatory increase in MKK6 protein levels. The reduced levels of p38 MAPK signalling in the diabetic kidneys of Mkk3 ( -/- ) db/db mice was associated with protection against the following: declining renal function, increasing albuminuria, renal hypertrophy, podocyte loss, mesangial cell activation and glomerular fibrosis. Diabetic Mkk3 ( -/- ) db/db mice were also significantly protected from tubular injury and interstitial fibrosis, which was associated with reduced Ccl2 mRNA expression and interstitial macrophage accumulation. CONCLUSIONS/INTERPRETATION: MKK3-p38 MAPK signalling is not required for the development of obesity or type 2 diabetes, but plays a distinct pathogenic role in the progression of diabetic nephropathy in db/db mice.

Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus.

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.

Correction.

The following sentence was omitted from the acknowledgment section of our report "Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms" (3 Feb., p. 682)(1) because of an error. "A. Lin and M. Karin are acknowledged for informing us abut the presence of an upstream in-frame initiation codon in the sequence of human MKK4/JNKK/SEK1 before publication."

Transcription factor ATF2 regulation by the JNK signal transduction pathway.

Treatment of cells with pro-inflammatory cytokines or ultraviolet radiation causes activation of the c-Jun NH2-terminal protein kinase (JNK). Activating transcription factor-2 (ATF2) was found to be a target of the JNK signal transduction pathway. ATF2 was phosphorylated by JNK on two closely spaced threonine residues within the NH2-terminal activation domain. The replacement of these phosphorylation sites with alanine inhibited the transcriptional activity of ATF2. These mutations also inhibited ATF2-stimulated gene expression mediated by the retinoblastoma (Rb) tumor suppressor and the adenovirus early region 1A (E1A) oncoprotein. Furthermore, expression of dominant-negative JNK inhibited ATF2 transcriptional activity. Together, these data demonstrate a role for the JNK signal transduction pathway in transcriptional responses mediated by ATF2.

An osmosensing signal transduction pathway in mammalian cells.

The osmotic balance between the cytoplasmic and extracellular compartments of cells is critical for the control of cell volume. A mammalian protein kinase, Jnk, which is a distant relative of the mitogen-activated protein kinase group, was activated by phosphorylation on threonine and tyrosine in osmotically shocked cells. The activation of Jnk may be relevant to the biological response to osmotic shock because the expression of human Jnk in the yeast Saccharomyces cerevisiae rescued a defect in growth on hyper-osmolar media. These data indicate that related protein kinases may mediate osmosensing signal transduction in yeast and mammalian cells.

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.

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.

Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.

Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.

Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway.

The nuclear factor of activated T cells (NFAT) group of transcription factors is retained in the cytoplasm of quiescent cells. NFAT activation is mediated in part by induced nuclear import. This process requires calcium-dependent dephosphorylation of NFAT caused by the phosphatase calcineurin. The c-Jun amino-terminal kinase (JNK) phosphorylates NFAT4 on two sites. Mutational removal of the JNK phosphorylation sites caused constitutive nuclear localization of NFAT4. In contrast, JNK activation in calcineurin-stimulated cells caused nuclear exclusion of NFAT4. These findings show that the nuclear accumulation of NFAT4 promoted by calcineurin is opposed by the JNK signal transduction pathway.

Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms.

Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms did not activate the ERK subgroup of MAP kinases, but MKK4 did activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways.

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.