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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.

A star is reborn In February this year the recurrent nova RS Ophiuchi (RS Oph) burst into life. Every 20 years or so the white dwarf component of this binary accumulates sufficient material from its red giant companion to power a thermonuclear explosion that we see as an increase in magnitude from a very dim 12.5 to magnitude 5. Two groups report observations of the recent outburst. Satellite X-ray observations by Sokoloski et al . reveal an initial phase in which the blast wave expanded freely. Within two days the outbound wave started to slow, suggesting that there was much less debris than had been expected from such an event. O'Brien et al . trained the largest terrestrial radio telescope arrays on RS Oph and were able to directly image a shock wave in a nova explosion for the first time, 14 days after its initial discovery. The structures revealed show an evolution to a remnant similar to that of a type II supernova — but evolving over months rather than millennia. Nova outbursts 1 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 2 , 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 3 , models of the previous (1985) outburst of RS Ophiuchi failed to adequately fit the X-ray evolution 4 and there was controversy over a single-epoch high-resolution radio image, which suggested that the remnant was bipolar 5 , 6 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 7 . 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.

We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated Bim EL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the Bim EL triple phosphomutant S100A/T112A/S114A instead of wild-type Bim EL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated Bim EL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-x L and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated Bim EL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.

Changes in protein phosphorylation represent a mechanism that is frequently employed by cells to regulate transcription factor activity. In response to alterations in the extracellular environment, signal transduction pathways target transcription factors, transcriptional coregulators and chromatin-modifying factors, leading to their phosphorylation by protein kinases or dephosphorylation by protein phosphatases. These modifications either positively or negatively regulate transcription factor activity to facilitate a program of gene expression that results in appropriate changes in cell behavior. Protein phosphorylation and dephosphorylation can directly regulate distinct aspects of transcription factor function, including cellular localization, protein stability, protein-protein interactions and DNA binding. The phosphorylation-dependent modulation of the activities of transcriptional coregulators and chromatin-modifying factors can also control transcription factor activity. Here we review recent studies that have led to a better understanding of the mechanisms by which protein phosphorylation and dephosphorylation governs transcription factor function.

Background: Acute hepatic failure secondary to paracetamol poisoning is associated with high mortality. C-jun (NH2) terminal kinase (JNK) is a member of the mitogen-activated protein kinase family and is a key intracellular signalling molecule involved in controlling the fate of cells. Aim: To examine the role of JNK in paracetamol-induced acute liver failure (ALF). Methods: A previously developed mouse model of paracetamol poisoning was used to examine the role of JNK in paracetamol-induced ALF. Results: Paracetamol-induced hepatic JNK activation both in human and murine paracetamol hepatotoxicity and in our murine model preceded the onset of hepatocyte death. JNK inhibition in vivo (using two JNK inhibitors with different mechanisms of action) markedly reduced mortality in murine paracetamol hepatotoxicity, with a significant reduction in hepatic necrosis and apoptosis. In addition, delayed administration of the JNK inhibitor was more effective than N-acetylcysteine after paracetamol poisoning in mice. JNK inhibition was not protective in acute carbon tetrachloride-mediated or anti-Fas antibody-mediated hepatic injury, suggesting specificity for the role of JNK in paracetamol hepatotoxicity. Furthermore, disruption of the JNK1 or JNK2 genes did not protect against paracetamol-induced hepatic damage. Pharmacological JNK inhibition had no effect on paracetamol metabolism, but markedly inhibited hepatic tumour necrosis foctor α (TNF α) production after paracetamol poisoning. Conclusions: These data demonstrated a central role for JNK in the pathogenesis of paracetamol-induced liver failure, thereby identifying JNK as an important therapeutic target in the treatment of paracetamol hepatotoxicity.

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.

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 NH$_2$-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.

The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to ultraviolet (UV) radiation. However, the functional consequence of JNK activation in UV-irradiated cells has not been established. It is shown here that JNK is required for UV-induced apoptosis in primary murine embryonic fibroblasts. Fibroblasts with simultaneous targeted disruptions of all the functional Jnk genes were protected against UV-stimulated apoptosis. The absence of JNK caused a defect in the mitochondrial death signaling pathway, including the failure to release cytochrome c. These data indicate that mitochondria are influenced by proapoptotic signal transduction through the JNK pathway.

Atomoxetine has been approved by the FDA as the first new drug in 30 years for the treatment of attention deficit/hyperactivity disorder (ADHD). As a selective norepinephrine uptake inhibitor and a nonstimulant, atomoxetine has a different mechanism of action from the stimulant drugs used up to now for the treatment of ADHD. Since brain acetylcholine (ACh) has been associated with memory, attention and motivation, processes dysregulated in ADHD, we investigated the effects of atomoxetine on cholinergic neurotransmission. We showed here that, in rats, atomoxetine (0.3–3 mg/kg, i.p.), – increases in vivo extracellular levels of ACh in cortical but not subcortical brain regions. The marked increase of cortical ACh induced by atomoxetine was dependent upon norepinephrine α -1 and/or dopamine D1 receptor activation. We observed similar increases in cortical and hippocampal ACh release with methylphenidate (1 and 3 mg/kg, i.p.) – currently the most commonly prescribed medication for the treatment of ADHD – and with the norepinephrine uptake inhibitor reboxetine (3–30 mg/kg, i.p.). Since drugs that increase cholinergic neurotransmission are used in the treatment of cognitive dysfunction and dementias, we also investigated the effects of atomoxetine on memory tasks. We showed that, consistent with its cortical procholinergic and catecholamine-enhancing profile, atomoxetine (1–3 mg/kg, p.o.) significantly ameliorated performance in the object recognition test and the radial arm-maze test.

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim EL , Bim L and Bim S ) are generated by alternative splicing. Bim EL , the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim EL through the DEF2 domain of Bim EL and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim EL degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim EL and Bim L are interchangeable, and that Bim S is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim EL .