Paralogous translation factors target distinct mRNAs to differentially regulate tolerance to oxidative stress in yeast.

Translation initiation factor 4G (eIF4G) is an integral component of the eIF4F complex which is key to translation initiation for most eukaryotic mRNAs. Many eIF4G isoforms have been described in diverse eukaryotic organisms but we currently have a poor understanding of their functional roles and whether they regulate translation in an mRNA specific manner. The yeast Saccharomyces cerevisiae expresses two eIF4G isoforms, eIF4G1 and eIF4G2, that have previously been considered as functionally redundant with any phenotypic differences arising due to alteration in eIF4G expression levels. Using homogenic strains that express eIF4G1 or eIF4G2 as the sole eIF4G isoforms at comparable expression levels to total eIF4G, we show that eIF4G1 is specifically required to mediate the translational response to oxidative stress. eIF4G1 binds the mRNA cap and remains associated with actively translating ribosomes during oxidative stress conditions and we use quantitative proteomics to show that eIF4G1 promotes oxidative stress-specific proteome changes. eIF4G1, but not eIF4G2, binds the Slf1 LARP protein which appears to mediate the eIF4G1-dependent translational response to oxidative stress. We show similar isoform specific roles for eIF4G in human cells suggesting convergent evolution of multiple eIF4G isoforms offers significant advantages especially where translation must continue under stress conditions.

Discovery of a Gatekeeper Residue in the C-Terminal Tail of the Extracellular Signal-Regulated Protein Kinase 5 (ERK5).

The extracellular signal-regulated protein kinase 5 (ERK5) is a non-redundant mitogen-activated protein kinase (MAPK) that exhibits a unique C-terminal extension which comprises distinct structural and functional properties. Here, we sought to elucidate the significance of phosphoacceptor sites in the C-terminal transactivation domain of ERK5. We have found that Thr732 acted as a functional gatekeeper residue controlling C-terminal-mediated nuclear translocation and transcriptional enhancement. Consistently, using a non-bias quantitative mass spectrometry approach, we demonstrated that phosphorylation at Thr732 conferred selectivity for binding interactions of ERK5 with proteins related to chromatin and RNA biology, whereas a number of metabolic regulators were associated with full-length wild type ERK5. Additionally, our proteomic analysis revealed that phosphorylation of the Ser730-Glu-Thr732-Pro motif could occur independently of dual phosphorylation at Thr218-Glu-Tyr220 in the activation loop. Collectively, our results firmly establish the significance of C-terminal phosphorylation in regulating ERK5 function. The post-translational modification of ERK5 on its C-terminal tail might be of particular relevance in cancer cells where ERK5 has be found to be hyperphosphoryated.

Mitochondrial Proteins Moonlighting in the Nucleus.

Mitochondria function as cellular energy generators, producing the fuel required to drive biological processes. The response of cells to mitochondrial activity or dysfunction regulates their survival, growth, proliferation, and differentiation. Several proteins that contain mitochondrial-targeting sequences (MTS) also reside in the nucleus and there is increasing evidence that the nuclear translocation of mitochondrial proteins represents a novel pathway by which mitochondria signal their status to the cell. Here, we discuss the different mechanisms that control the dual mitochondrial and nuclear localisation of proteins and propose that these nuclear moonlighters represent a widespread regulatory circuit to maintain mitochondrial homeostasis.

Regulation of axon growth by the JIP1-AKT axis.

The polarisation of developing neurons to form axons and dendrites is required for the establishment of neuronal connections leading to proper brain function. The protein kinase AKT and the MAP kinase scaffold protein JNK-interacting protein-1 (JIP1) are important regulators of axon formation. Here we report that JIP1 and AKT colocalise in axonal growth cones of cortical neurons and collaborate to promote axon growth. The loss of AKT protein from the growth cone results in the degradation of JIP1 by the proteasome, and the loss of JIP1 promotes a similar fate for AKT. Reduced protein levels of both JIP1 and AKT in the growth cone can be induced by glutamate and this coincides with reduced axon growth, which can be rescued by a stabilized mutant of JIP1 that rescues AKT protein levels. Taken together, our data reveal a collaborative relationship between JIP1 and AKT that is required for axon growth and can be regulated by changes in neuronal activity.

Ribonucleoprotein bodies are phased in.

Intracellular compartments are necessary for the regulation of many biochemical processes that ensure cell survival, growth and proliferation. Compartmentalisation is commonly achieved in organelles with defined lipid membranes, such as mitochondria, endoplasmic reticulum or the Golgi apparatus. While these organelles are responsible for many localised biochemical processes, recent evidence points to another class of compartments that lack membrane boundaries. The structure and content of these bodies depend on their function and subcellular localisation, but they mainly incorporate proteins and RNA. Examples of these ribonucleoprotein bodies (RNPBs) include eukaryotic mRNA processing bodies (P-bodies) and stress granules (SGs). While most of these structures have been widely studied for their capacity to bind, store and process mRNAs under different conditions, their biological functions and physical properties are poorly understood. Recent intriguing data suggest that liquid-liquid phase separation (LLPS) represents an important mechanism seeding the formation and defining the function of RNPBs. In this review, we discuss how LLPS is transforming our ideas about the biological functions of SGs and P-bodies and their link to diseases.

Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch.

The epidermis is a highly organized structure, the integrity of which is central to the protection of an organism. Development and subsequent maintenance of this tissue depends critically on the intricate balance between proliferation and differentiation of a resident stem cell population; however, the signals controlling the proliferation-differentiation switch in vivo remain elusive. Here, we show that mice carrying a homozygous missense mutation in interferon regulatory factor 6 (Irf6), the homolog of the gene mutated in the human congenital disorders Van der Woude syndrome and popliteal pterygium syndrome, have a hyperproliferative epidermis that fails to undergo terminal differentiation, resulting in soft tissue fusions. We further demonstrate that mice that are compound heterozygotes for mutations in Irf6 and the gene encoding the cell cycle regulator protein stratifin (Sfn; also known as 14-3-3sigma) show similar defects of keratinizing epithelia. Our results indicate that Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch and that Irf6 and Sfn interact genetically in this process.

Docking interactions of the JNK scaffold protein WDR62.

JNK (c-Jun N-terminal kinase) is part of a MAPK (mitogen-activated protein kinase) signalling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signalling pathway and play a crucial role in signal transmission and MAPK regulation. WDR62 (WD repeat domain 62) is a JNK scaffold protein. Recessive mutations within WDR62 result in severe cerebral cortical malformation. In the present study we demonstrate the association of WDR62 with endogenous and overexpressed proteins of both JNK2 and the JNK2-activating kinase MKK7 (MAPK kinase 7). Association of WDR62 with JNK2 and MKK7 occurs via direct protein-protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a D domain motif located at the C-terminus. A WDR62 mutant lacking the putative JNK-binding domain fails to activate and recruit JNK to cellular granules. Furthermore, a synthetic peptide composed of the WDR62 docking domain inhibits JNK2 activity in vitro. WDR62 association with JNK2 requires both the JNK CD and ED domains, and the binding requisite is distinct from that of the previously described JNK2 association with JIP1 (JNK-interacting protein 1). Next, we characterized the association between WDR62 and MKK7. WDR62 associates directly with the MKK7ß1 isoform independently of JNK binding, but fails to interact with MKK7α1. Furthermore, MKK7ß1 recruits a protein phosphatase that dephosphorylates WDR62. Interestingly, a premature termination mutation in WDR62 that results in severe brain developmental defects does not abrogate WDR62 association with either JNK or MKK7. Therefore such mutations represent a loss of WDR62 function independent of JNK signalling.

Inhibiting ERK5 overcomes breast cancer resistance to anti-HER2 therapy by targeting the G1/S cell cycle transition.

Targeting the human epidermal growth factor receptor 2 (HER2) became a landmark in the treatment of HER2-driven breast cancer. Nonetheless, the clinical efficacy of anti-HER2 therapies can be short-lived and a significant proportion of patients ultimately develop metastatic disease and die. One striking consequence of oncogenic activation of HER2 in breast cancer cells is the constitutive activation of the extracellular-regulated protein kinase 5 (ERK5) through its hyperphosphorylation. In this study, we sought to decipher the significance of this unique molecular signature in promoting therapeutic resistance to anti-HER2 agents. We found that a small-molecule inhibitor of ERK5 suppressed the phosphorylation of the retinoblastoma protein (RB) in HER2 positive breast cancer cells. As a result, ERK5 inhibition enhanced the anti-proliferative activity of single-agent anti-HER2 therapy in resistant breast cancer cell lines by causing a G1 cell cycle arrest. Moreover, ERK5 knockdown restored the anti-tumor activity of the anti-HER2 agent lapatinib in human breast cancer xenografts. Taken together, these findings support the therapeutic potential of ERK5 inhibitors to improve the clinical benefit that patients receive from targeted HER2 therapies.

The JIP1 scaffold protein regulates axonal development in cortical neurons.

The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling.JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1-4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification,it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.

A central role for p38 MAPK in the early transcriptional response to stress.

The mitogen-activated protein kinase p38 (p38 MAPK) is activated by a number of stresses. A recent study in BMC Genomics has uncovered the early transcriptional responses to three types of stress and has demonstrated a central role for p38 MAPK in mediating these responses. See research article http://www.biomedcentral.com/1471-2164/11/144.

Correction to: The mTOR-S6 kinase pathway promotes stress granule assembly.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of gene transcription by mitogen-activated protein kinase signaling pathways.

Mitogen-activated protein kinase (MAPK) signaling pathways are key mediators of eukaryotic transcriptional responses to extracellular signals. These pathways control gene expression in a number of ways including the phosphorylation and regulation of transcription factors, co-regulatory proteins and chromatin proteins. MAPK pathways therefore target multiple components of transcriptional complexes at gene promoters and can regulate DNA binding, protein stability, cellular localization, transactivation or repression, and nucleosome structure. Recent work has uncovered further complexities in the mechanisms by which MAPKs control gene expression including their roles as integral components of transcription factor complexes and their interplay with other post-translational modification pathways. In this review I discuss these advances with particular focus on how MAPK signals are integrated by transcription factor complexes to provide specific transcriptional responses and how this relates to cellular function.

Regulation of p73-mediated apoptosis by c-Jun N-terminal kinase.

The JNK (c-Jun N-terminal kinase)/mitogen-activated protein kinase signalling pathway is a major mediator of stress responses in cells, including the response to DNA damage. DNA damage also causes the stabilization and activation of p73, a member of the p53 family of transcription factors. p73, like p53, can mediate apoptosis by up-regulating the expression of pro-apoptotic genes, including Bax (Bcl2-associated X protein) and PUMA (p53 up-regulated modulator of apoptosis). Changes in p73 expression have been linked to tumour progression, particularly in neuroblastomas, whereas in tumours that feature inactivated p53 there is evidence that p73 may mediate the apoptotic response to chemotherapeutic agents. In the present study, we demonstrate a novel link between the JNK signalling pathway and p73. We use pharmacological and genetic approaches to show that JNK is required for p73-mediated apoptosis induced by the DNA damaging agent cisplatin. JNK forms a complex with p73 and phosphorylates it at several serine and threonine residues. The mutation of JNK phosphorylation sites in p73 abrogates cisplatin-induced stabilization of p73 protein, leading to a reduction in p73 transcriptional activity and reduced p73-mediated apoptosis. Our results demonstrate that the JNK pathway is an important regulator of DNA damage-induced apoptosis mediated by p73.

The mTOR-S6 kinase pathway promotes stress granule assembly.

Stress granules are cytoplasmic mRNA-protein complexes that form upon the inhibition of translation initiation and promote cell survival in response to environmental insults. However, they are often associated with pathologies, including neurodegeneration and cancer, and changes in their dynamics are implicated in ageing. Here we show that the mTOR effector kinases S6 kinase 1 (S6K1) and S6 kinase 2 (S6K2) localise to stress granules in human cells and are required for their assembly and maintenance after mild oxidative stress. The roles of S6K1 and S6K2 are distinct, with S6K1 having a more significant role in the formation of stress granules via the regulation of eIF2α phosphorylation, while S6K2 is important for their persistence. In C. elegans, the S6 kinase orthologue RSKS-1 promotes the assembly of stress granules and its loss of function sensitises the nematodes to stress-induced death. This study identifies S6 kinases as regulators of stress granule dynamics and provides a novel link between mTOR signalling, translation inhibition and survival.

A new role for an old enemy.

Drugs that change the shape of AKT, a protein kinase that promotes tumor growth, may be more effective than drugs that only target its enzymatic activity.

A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity.

The coordinated regulation of mitochondrial and nuclear activities is essential for cellular respiration and its disruption leads to mitochondrial dysfunction, a hallmark of ageing. Mitochondria communicate with nuclei through retrograde signalling pathways that modulate nuclear gene expression to maintain mitochondrial homeostasis. The monooxygenase CLK-1 (human homologue COQ7) was previously reported to be mitochondrial, with a role in respiration and longevity. We have uncovered a distinct nuclear form of CLK-1 that independently regulates lifespan. Nuclear CLK-1 mediates a retrograde signalling pathway that is conserved from Caenorhabditis elegans to humans and is responsive to mitochondrial reactive oxygen species, thus acting as a barometer of oxidative metabolism. We show that, through modulation of gene expression, the pathway regulates both mitochondrial reactive oxygen species metabolism and the mitochondrial unfolded protein response. Our results demonstrate that a respiratory enzyme acts in the nucleus to control mitochondrial stress responses and longevity.

Transcriptional regulation by the MAP kinase signaling cascades.

A major function of the mitogen-activated protein kinase (MAPK) pathways is to control eukaryotic gene expression programmes in response to extracellular signals. MAPKs directly control gene expression by phosphorylating transcription factors. However, it is becoming clear that transcriptional regulation in response to MAPK signaling is more complex. MAPKs can also target coactivators and corepressors and affect nucleosomal structure by inducing histone modifications. Furthermore, multiple inputs into individual promoters can be elicited by MAPKs by targeting different components of the same coregulatory complex or by triggering different events on the same transcription factor. "Postgenomic approaches" are beginning to impact on our understanding of these gene regulatory networks. In this review, we summarise the current knowledge of MAPK-mediated gene regulation, and focus on how complexities in signaling outcomes are achieved and how this relates to physiological processes.

Targeting apoptosis signalling kinase-1 (ASK-1) does not prevent the development of neuropathy in streptozotocin-induced diabetic mice.

Apoptosis signal-regulating kinase-1 (ASK1) is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K) which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n) mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy.

A new regulator of caveolae signalling.

Cavin-3 regulates metabolism and cell proliferation by coordinating the activities of growth factor signalling cascades.