A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.

Active p38α causes macrovesicular fatty liver in mice.

One third of the western population suffers from nonalcoholic fatty liver disease (NAFLD), which may ultimately develop into hepatocellular carcinoma (HCC). The molecular event(s) that triggers the disease are not clear. Current understanding, known as the multiple hits model, suggests that NAFLD is a result of diverse events at several tissues (e.g., liver, adipose tissues, and intestine) combined with changes in metabolism and microbiome. In contrast to this prevailing concept, we report that fatty liver could be triggered by a single mutated protein expressed only in the liver. We established a transgenic system that allows temporally controlled activation of the MAP kinase p38α in a tissue-specific manner by induced expression of intrinsically active p38α allele. Here we checked the effect of exclusive activation in the liver. Unexpectedly, induction of p38α alone was sufficient to cause macrovesicular fatty liver. Animals did not become overweight, showing that fatty liver can be imposed solely by a genetic modification in liver per se and can be separated from obesity. Active p38α-induced fatty liver is associated with up-regulation of MUC13, CIDEA, PPARγ, ATF3, and c-jun mRNAs, which are up-regulated in human HCC. Shutting off expression of the p38α mutant resulted in reversal of symptoms. The findings suggest that p38α plays a direct causative role in fatty liver diseases and perhaps in other chronic inflammatory diseases. As p38α activity was induced by point mutations, it could be considered a proto-inflammatory gene (proto-inflammagene).

Asynchronous Pattern of MAPKs' Activity during Aging of Different Tissues and of Distinct Types of Skeletal Muscle.

The MAPK p38α was proposed to be a prominent promoter of skeletal muscle aging. The skeletal muscle tissue is composed of various muscle types, and it is not known if p38α is associated with aging in all of them. It is also not known if p38α is associated with aging of other tissues. JNK and ERK were also proposed to be associated with aging of several tissues. Nevertheless, the pattern of p38α, JNK, and ERK activity during aging was not documented. Here, we documented the levels of phosphorylated/active p38α, Erk1/2, and JNKs in several organs as well as the soleus, tibialis anterior, quadriceps, gastrocnemius, and EDL muscles of 1-, 3-, 6-, 13-, 18-, and 24-month-old mice. We report that in most tissues and skeletal muscles, the MAPKs' activity does not change in the course of aging. In most tissues and muscles, p38α is in fact active at younger ages. The quadriceps and the lungs are exceptions, where p38α is significantly active only in mice 13 months old or older. Curiously, levels of active JNK and ERKs are also elevated in aged lungs and quadriceps. RNA-seq analysis of the quadriceps during aging revealed downregulation of proteins related to the extra-cellular matrix (ECM) and ERK signaling. A panel of mRNAs encoding cell cycle inhibitors and senescence-associated proteins, considered to be aging markers, was not found to be elevated. It seems that the pattern of MAPKs' activation in aging, as well as expression of known 'aging' components, are tissue- and muscle type-specific, supporting a notion that the process of aging is tissue- and even cell-specific.

Differential Modulation of the Phosphoproteome by the MAP Kinases Isoforms p38α and p38ß.

The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38ß are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38ß, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38αWT, p38ßWT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α-/-, and p38ß-/- MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38ß suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38ß signaling and on the specific targets of these two kinases.

How Do Protein Kinases Take a Selfie (Autophosphorylate)?

Eukaryotic protein kinases (EPKs) control most biological processes and play central roles in many human diseases. To become catalytically active, EPKs undergo conversion from an inactive to an active conformation, an event that depends upon phosphorylation of their activation loop. Intriguingly, EPKs can use their own catalytic activity to achieve this critical phosphorylation. In other words, paradoxically, EPKs catalyze autophosphorylation when supposedly in their inactive state. This indicates the existence of another important conformation that specifically permits autophosphorylation at the activation loop, which in turn imposes adoption of the active conformation. This can be considered a prone-to-autophosphorylate conformation. Recent findings suggest that in prone-to-autophosphorylate conformations catalytic motifs are aligned allosterically, by dimerization or by regulators, and support autophosphorylation in cis or trans.

The p38ß mitogen-activated protein kinase possesses an intrinsic autophosphorylation activity, generated by a short region composed of the α-G helix and MAPK insert.

Protein kinases are regulated by a large number of mechanisms that vary from one kinase to another. However, a fundamental activation mechanism shared by all protein kinases is phosphorylation of a conserved activation loop threonine residue. This is achieved in many cases via autophosphorylation. The mechanism and structural basis for autophosphorylation are not clear and are in fact enigmatic because this phosphorylation occurs when the kinase is in its inactive conformation. Unlike most protein kinases, MAP kinases are not commonly activated by autophosphorylation but rather by MEK-dependent phosphorylation. Here we show that p38ß, a p38 isoform that is almost identical to p38α, is exceptional and spontaneously autoactivates by autophosphorylation. We identified a 13-residue-long region composed of part of the αG-helix and the MAPK insert that triggers the intrinsic autophosphorylation activity of p38ß. When inserted into p38α, this fragment renders it spontaneously active in vitro and in mammalian cells. We further found that an interaction between the N terminus and a particular region of the C-terminal extension suppresses the intrinsic autophosphorylation of p38ß in mammalian cells. Thus, this study identified the structural motif responsible for the unique autophosphorylation capability of p38ß and the motif inhibiting this activity in living cells. It shows that the MAPK insert and C-terminal extension, structural motifs that are unique to MAPKs, play a critical role in controlling autophosphorylation.

DEF pocket in p38α facilitates substrate selectivity and mediates autophosphorylation.

Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α.

The ER-Golgi transport of influenza virus through NS1-Sec13 association during virus replication.

IMPORTANCE: Influenza A virus is a respiratory virus that can cause complications such as acute bronchitis and secondary bacterial pneumonia. Drug therapies and vaccines are available against influenza, albeit limited by drug resistance and the non-universal vaccine administration. Hence there is a need for host-targeted therapies against influenza to provide an effective alternative therapeutic target. Sec13 was identified as a novel host interactor of influenza. Endoplasmic reticulum-to-Golgi transport is an important pathway of influenza virus replication and viral export. Specifically, Sec13 has a functional role in influenza replication and virulence.

Overexpression of PDE2 or SSD1-V in Saccharomyces cerevisiae W303-1A strain renders it ethanol-tolerant.

Understanding the genetic basis of the yeast ability to proliferate and ferment in the presence of restrictive concentrations of ethanol is of importance to both science and technology. In this study, we searched for genes that improve ethanol tolerance in ethanol-sensitive strains. To screen for suppressors of ethanol sensitivity, we introduced a 2µ-based genomic library, prepared from the ethanol-tolerant yeast S288C, into the ethanol-sensitive strain W303-1A. Two genomic fragments from this library rescued the ethanol sensitivity of W303-1A. One contained the PDE2 gene, which when over-expressed, conferred ethanol tolerance. Surprisingly, the effect of PDE2 was not mediated via MSN2/MSN4 transcription factors, as it was able to improve ethanol tolerance in msn2Δmsn4Δ strain. In the second genomic fragment, it was the N-terminal region of the SSD1 gene that carried the ethanol-tolerant phenotype. The SSD1-V allele of the polymorphic SSD1 gene expressed from a low-copy number plasmid also resulted in the tolerant phenotype. Both SSD1 and PDE2 seemed to improve ethanol tolerance by maintaining robustness of the yeast cell wall.

Turbidostat culture of Saccharomyces cerevisiae W303-1A under selective pressure elicited by ethanol selects for mutations in SSD1 and UTH1.

We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303-1A under the selective pressure of ethanol. W303-1A was subjected to three rounds of turbidostat, in a medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 to 0.32 h(-1) . Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within 6 days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within 2 days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of six clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 of 19 tolerant clones, the stop codon in ssd1-d was replaced with an amino acid-encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol.

A Review and Meta-Analysis of Influenza Interactome Studies.

Annually, the influenza virus causes 500,000 deaths worldwide. Influenza-associated mortality and morbidity is especially high among the elderly, children, and patients with chronic diseases. While there are antivirals available against influenza, such as neuraminidase inhibitors and adamantanes, there is growing resistance against these drugs. Thus, there is a need for novel antivirals for resistant influenza strains. Host-directed therapies are a potential strategy for influenza as host processes are conserved and are less prone mutations as compared to virus-directed therapies. A literature search was performed for papers that performed viral-host interaction screens and the Reactome pathway database was used for the bioinformatics analysis. A total of 15 studies were curated and 1717 common interactors were uncovered among all these studies. KEGG analysis, Enrichr analysis, STRING interaction analysis was performed on these interactors. Therefore, we have identified novel host pathways that can be targeted for host-directed therapy against influenza in our review.

Targeting the p38α pathway in chronic inflammatory diseases: Could activation, not inhibition, be the appropriate therapeutic strategy?

Chronic inflammatory diseases (CIDs) afflict millions worldwide and remain incurable. The mitogen-activated protein kinase (MAPK) p38α is a critical node in the intricate acute inflammatory response. It induces the production of various pro-inflammatory mediators, primarily via the MAPK-activated protein kinase 2 (MK2). This, coupled with its sustained activation in CIDs, has led to the assumption that dysregulated pro-inflammatory p38α-dependent pathways are central drivers of chronic inflammation. Inhibiting the p38α cascade thus seems a logical therapeutic strategy, leading to significant efforts towards developing p38α- and MK2-specific inhibitors. However, recent studies raise the possibility that the effects of chronic p38α activation in CIDs have been misinterpreted. In cell cultures and murine models, constitutive p38α activity causes dramatic downregulation, rather than activation, of downstream elements such as MK2, via the ubiquitin-proteasome system, and phospho-Hsp27. Perhaps, sustained p38α activity promotes CIDs by inducing degradation of essential components of the p38α pathway. If this notion is genuine, then the current pharmacological strategy, focused on the inhibition of these components, is counter-productive and may explain why no p38α or MK2 inhibitor has made it to the clinic. It could be that an appropriate strategy should involve restoring or inducing certain p38α targets instead.

SSeCKS/Gravin/AKAP12 inhibits cancer cell invasiveness and chemotaxis by suppressing a protein kinase C- Raf/MEK/ERK pathway.

SSeCKS/Gravin/AKAP12 ("SSeCKS") encodes a cytoskeletal protein that regulates G(1) --> S progression by scaffolding cyclins, protein kinase C (PKC) and PKA. SSeCKS is down-regulated in many tumor types including prostate, and when re-expressed in MAT-LyLu (MLL) prostate cancer cells, SSeCKS selectively inhibits metastasis by suppressing neovascularization at distal sites, correlating with its ability to down-regulate proangiogenic genes including Vegfa. However, the forced re-expression of VEGF only rescues partial lung metastasis formation. Here, we show that SSeCKS potently inhibits chemotaxis and Matrigel invasion, motility parameters contributing to metastasis formation. SSeCKS suppressed serum-induced activation of the Raf/MEK/ERK pathway, resulting in down-regulation of matrix metalloproteinase-2 expression. In contrast, SSeCKS had no effect on serum-induced phosphorylation of the Src substrate, Shc, in agreement with our previous data that SSeCKS does not inhibit Src kinase activity in cells. Invasiveness and chemotaxis could be restored by the forced expression of constitutively active MEK1, MEK2, ERK1, or PKCalpha. SSeCKS suppressed phorbol ester-induced ERK1/2 activity only if it encoded its PKC binding domain (amino acids 553-900), suggesting that SSeCKS attenuates ERK activation through a direct scaffolding of conventional and/or novel PKC isozymes. Finally, control of MLL invasiveness by SSeCKS is influenced by the actin cytoskeleton: the ability of SSeCKS to inhibit podosome formation is unaffected by cytochalasin D or jasplakinolide, whereas its ability to inhibit MEK1/2 and ERK1/2 activation is nullified by jasplakinolide. Our findings suggest that SSeCKS suppresses metastatic motility by disengaging activated Src and then inhibiting the PKC-Raf/MEK/ERK pathways controlling matrix metalloproteinase-2 expression and podosome formation.

Conformational bias imposed by source microseeds results in structural ambiguity.

The p38 MAP kinase pathway is an essential component of numerous cellular signalling networks which are usually activated in response to extracellular environmental stress conditions. In addition to the canonical activation, several alternative activation pathways have been identified for p38; one of these, in which p38 is initially phosphorylated on Tyr323 and consequently autoactivated, is exclusive to T cells and is induced by TCR activation. Intrinsically active and inactive mutants at position 323 have been developed in order to evaluate the structural changes that occur upon TCR-induced activation. In order to promote crystal growth, cross streak-seeding techniques were utilized. This technique has gained popularity in promoting crystal growth when spontaneous nucleation induces critical defects or is being entirely hindered. The crystal characteristics of some mutants were highly similar to those of the wild-type source seeds (form A). In contrast, other mutants crystallized spontaneously with a different space group and molecular packing (form B). One of the active mutants (Y323T) crystallized in both crystal forms, displaying different packing characteristics and significant differences in molecular conformation that were clearly dictated by the source seeds. This implies that the source seeds used in cross streak-seeding could, in some cases, impose bias on the structural outcome of the studied molecule. Such incidents could occur when the conformational freedom permits crystal packing while not reflecting the authentic structure.

Expression of a constitutively active p38α mutant in mice causes early death, anemia, and accumulation of immunosuppressive cells.

The MAP kinase p38α is associated with numerous processes in eukaryotes, and its elevated activity is a prominent feature of inflammatory diseases, allergies, and aging. Since p38α is a nodal component of a complex signaling network, it is difficult to reveal exactly how p38α contributes to disparate outcomes. Identification of p38α -specific effects requires activation of p38α per se in vivo. We generated a transgenic mouse model that meets this requirement by allowing inducible and reversible expression of an intrinsically active p38α molecule (p38αD176A+F327S ). p38α's activation across all murine tissues resulted in a significant loss of body weight and death of about 40% of the mice within 17 weeks of activation, although most tissues were unaffected. Flow cytometric analysis of the lungs and bronchoalveolar lavage fluid detected an accumulation of 'debris' within the airways, suggesting impaired clearance. It also revealed increased numbers of alternatively activated alveolar macrophages and myeloid-derived suppressor cells within the lung, pointing at suppression and resolution of inflammation. Blood count suggested that mice expressing p38αD176A+F327S suffer from hemolytic anemia. Flow cytometry of bone marrow revealed a reduced number of hematopoietic stem cells and abnormalities in the erythroid lineage. Unexpectedly, p38α's substrate MAPKAPK2, mitogen-activated protein kinase-activated protein kinase 2 was downregulated in mice expressing p38αD176A+F327S , suggesting that constitutive activity of p38α may impose pathological phenotypes by downregulating downstream components, perhaps via a feedback inhibition mechanism. In summary, this new mouse model shows that induced p38α activity per se is hazardous to mouse vitality and welfare, although pathological parameters are apparent only in blood count, bone marrow, and lungs.

When expressed in yeast, mammalian mitogen-activated protein kinases lose proper regulation and become spontaneously phosphorylated.

MAPKs (mitogen-activated protein kinases) are key components in cell signalling pathways. Under optimal growth conditions, their activity is kept off, but in response to stimulation it is dramatically evoked. Because of the high degree of evolutionary conservation at the levels of sequence and mode of activation, MAPKs are believed to share similar regulatory mechanisms in all eukaryotes and to be functionally substitutable between them. To assess the reliability of this notion, we systematically analysed the activity, regulation and phenotypic effects of mammalian MAPKs in yeast. Unexpectedly, all mammalian MAPKs tested were spontaneously phosphorylated in yeast. JNKs (c-Jun N-terminal kinases) lost their phosphorylation in pbs2Delta cells, but p38s and ERKs (extracellular-signal-regulated kinases) maintained their spontaneous phosphorylation even in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Kinase-dead variants of ERKs and p38s were phosphorylated in strains lacking a single MEK (MAPK/ERK kinase), but not in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Thus, in yeast, p38 and ERKs are phosphorylated via a combined mechanism of autophosphorylation and MEK-mediated phosphorylation (any MEK). We further addressed the mechanism allowing mammalian MAPKs to exploit yeast MEKs in the absence of any activating signal. We suggest that mammalian MAPKs lost during evolution a C-terminal region that exists in some yeast MAPKs. Indeed, removal of this region from Hog1 and Mpk1 rendered them spontaneously and highly phosphorylated. It implies that MAPKs possess an efficient inherent autoposphorylation capability that is suppressed in yeast MAPKs via a C-terminal domain and in mammalian MAPKs via as yet unknown means.

Mutations That Confer Drug-Resistance, Oncogenicity and Intrinsic Activity on the ERK MAP Kinases-Current State of the Art.

Unique characteristics distinguish extracellular signal-regulated kinases (Erks) from other eukaryotic protein kinases (ePKs). Unlike most ePKs, Erks do not autoactivate and they manifest no basal activity; they become catalysts only when dually phosphorylated on neighboring Thr and Tyr residues and they possess unique structural motifs. Erks function as the sole targets of the receptor tyrosine kinases (RTKs)-Ras-Raf-MEK signaling cascade, which controls numerous physiological processes and is mutated in most cancers. Erks are therefore the executers of the pathway's biology and pathology. As oncogenic mutations have not been identified in Erks themselves, combined with the tight regulation of their activity, Erks have been considered immune against mutations that would render them intrinsically active. Nevertheless, several such mutations have been generated on the basis of structure-function analysis, understanding of ePK evolution and, mostly, via genetic screens in lower eukaryotes. One of the mutations conferred oncogenic properties on Erk1. The number of interesting mutations in Erks has dramatically increased following the development of Erk-specific pharmacological inhibitors and identification of mutations that cause resistance to these compounds. Several mutations have been recently identified in cancer patients. Here we summarize the mutations identified in Erks so far, describe their properties and discuss their possible mechanism of action.

Hog1-induced transcription of RTC3 and HSP12 is robust and occurs in cells lacking Msn2, Msn4, Hot1 and Sko1.

The yeast MAP kinase Hog1 pathway activates transcription of several hundreds genes. Large-scale gene expression and DNA binding assays suggest that most Hog1-induced genes are regulated by the transcriptional activators Msn2/4, Hot1 and Sko1. These studies also revealed the target genes of each activator and the putative binding sites on their promoters. In a previous study we identified a group of genes, which we considered the bona fide targets of Hog1, because they were induced in response to expression of intrinsically active mutant of Hog1, in the absence of any stress. We previously analyzed the promoter of the most highly induced gene, STL1, and noticed that some promoter properties were different from those proposed by large-scale data. We therefore continue to study promoters individually and present here analyses of promoters of more Hog1's targets, RTC3, HSP12, DAK1 and ALD3. We report that RTC3 and HSP12 promoters are robust and are induced, to different degrees, even in cells lacking all four activators. DAK1 and ALD3 promoters are not robust and fully depend on a single activator, DAK1 on Sko1 and ALD3 on Msn2/4. Most of these observations could not be inferred from the large-scale data. Msn2/4 are involved in regulating all four promoters. It was assumed, therefore, that the promoters are spontaneously active in ras2Δ cells, in which Msn2/4 are known to be de-repressed. Intriguingly, the promoters were not active in BY4741ras2Δ cells, but were de-repressed, as expected, in ras2Δ cells of other genetic backgrounds. This study describes two phenomena. One, some Hog1's target promoters are most robust, backupped by many activators. Second, in contrast to most laboratory strains, the widely used BY4741 strain does not induce Msn2/4 activity when the Ras/cAMP cascade is downregulated.

An Activating Mutation in ERK Causes Hyperplastic Tumors in a scribble Mutant Tissue in Drosophila.

Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf, or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo To determine if a point mutation could render ERK intrinsically active and oncogenic, we have assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog and has also been identified in a few human tumors. Our analyses indicate that a fly ERK ortholog harboring this mutation alone (RolledR80S), and more so in conjunction with the known sevenmaker mutation (RolledR80S+D334N), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of RolledR80S and RolledR80S+D334N induces tissue overgrowth in an established Drosophila cancer model. Our findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.