Suppression of mutant Kirsten-RAS (KRASG12D)-driven pancreatic carcinogenesis by dual-specificity MAP kinase phosphatases 5 and 6.

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRASG12D-driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRASG12D-driven pancreatic hyperplasia. This is accompanied by increased pancreatic acinar to ductal metaplasia (ADM) and the development of pre-neoplastic pancreatic intraepithelial neoplasia (PanINs). In contrast, by 100-days, pancreatic hyperplasia is reversed with significant atrophy of pancreatic tissue and weight loss observed in animals lacking either DUSP5 or DUSP6. On further ageing, Dusp6-/- mice display accelerated development of metastatic pancreatic ductal adenocarcinoma (PDAC), while in Dusp5-/- animals, although PDAC development is increased this process is attenuated by atrophy of pancreatic acinar tissue and severe weight loss in some animals before cancer could progress. Our data suggest that despite a common target in the ERK MAP kinase, DUSP5 and DUSP6 play partially non-redundant roles in suppressing oncogenic KRASG12D signalling, thus retarding both tumour initiation and progression. Our data suggest that loss of either DUSP5 or DUSP6, as observed in certain human tumours, including the pancreas, could promote carcinogenesis.

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe.

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.

Dual-specificity phosphatase 5 controls the localized inhibition, propagation, and transforming potential of ERK signaling.

Deregulated extracellular signal-regulated kinase (ERK) signaling drives cancer growth. Normally, ERK activity is self-limiting by the rapid inactivation of upstream kinases and delayed induction of dual-specificity MAP kinase phosphatases (MKPs/DUSPs). However, interactions between these feedback mechanisms are unclear. Here we show that, although the MKP DUSP5 both inactivates and anchors ERK in the nucleus, it paradoxically increases and prolongs cytoplasmic ERK activity. The latter effect is caused, at least in part, by the relief of ERK-mediated RAF inhibition. The importance of this spatiotemporal interaction between these distinct feedback mechanisms is illustrated by the fact that expression of oncogenic BRAFV600E, a feedback-insensitive mutant RAF kinase, reprograms DUSP5 into a cell-wide ERK inhibitor that facilitates cell proliferation and transformation. In contrast, DUSP5 deletion causes BRAFV600E-induced ERK hyperactivation and cellular senescence. Thus, feedback interactions within the ERK pathway can regulate cell proliferation and transformation, and suggest oncogene-specific roles for DUSP5 in controlling ERK signaling and cell fate.

Visualizing and Quantitating the Spatiotemporal Regulation of Ras/ERK Signaling by Dual-Specificity Mitogen-Activated Protein Phosphatases (MKPs).

The spatiotemporal regulation of the Ras/ERK pathway is critical in determining the physiological and pathophysiological outcome of signaling. Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (DUSPs or MKPs) are key regulators of pathway activity and may also localize ERK to distinct subcellular locations. Here we present methods largely based on the use of high content microscopy to both visualize and quantitate the subcellular distribution of activated (p-ERK) and total ERK in populations of mouse embryonic fibroblasts derived from mice lacking DUSP5, a nuclear ERK-specific MKP. Such methods in combination with rescue experiments using adenoviral vectors encoding wild-type and mutant forms of DUSP5 have allowed us to visualize specific defects in ERK regulation in these cells thus confirming the role of this phosphatase as both a nuclear regulator of ERK activity and localization.

Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases.

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK)-MEDIATED FEEDBACK LOOPS CONTROL HORMONE SENSING.

Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.

MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.

The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the 'gatekeepers' of ERK1/2 activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress.

Dual-specificity phosphatase 5 regulates nuclear ERK activity and suppresses skin cancer by inhibiting mutant Harvey-Ras (HRasQ61L)-driven SerpinB2 expression.

Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas(Q61L))-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5(-/-) mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5(+/+) cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas(Q61L) and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5(-/-), SerpinB2(-/-) double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.

CiteAb: a searchable antibody database that ranks antibodies by the number of times they have been cited.

BACKGROUND: Research antibodies are used by thousands of scientists working in diverse disciplines, but it is common to hear concerns about antibody quality. This means that researchers need to carefully choose the antibodies they use to avoid wasting time and money. A well accepted way of selecting a research antibody is to identify one which has been used previously, where the associated data has been peer-reviewed and the results published. DESCRIPTION: CiteAb is a searchable database which ranks antibodies by the number of times they have been cited. This allows researchers to easily find antibodies that have been used in peer-reviewed publications and the accompanying citations are listed, so users can check the data contained within the publications. This makes CiteAb a useful resource for identifying antibodies for experiments and also for finding information to demonstrate antibody validation. The database currently contains 1,400,000 antibodies which are from 90 suppliers, including 87 commercial companies and 3 academic resources. Associated with these antibodies are 140,000 publications which provide 306,000 antibody citations. In addition to searching, users can also browse through the antibodies and add their own publications to the CiteAb database. CONCLUSIONS: CiteAb provides a new way for researchers to find research antibodies that have been used successfully in peer-reviewed publications. It aims to assist these researchers and will hopefully help promote progress in many areas of life science research.

Signaling to extracellular signal-regulated kinase from ErbB1 kinase and protein kinase C: feedback, heterogeneity, and gating.

Many extracellular signals act via the Raf/MEK/ERK cascade in which kinetics, cell-cell variability, and sensitivity of the ERK response can all influence cell fate. Here we used automated microscopy to explore the effects of ERK-mediated negative feedback on these attributes in cells expressing endogenous ERK or ERK2-GFP reporters. We studied acute rather than chronic stimulation with either epidermal growth factor (ErbB1 activation) or phorbol 12,13-dibutyrate (PKC activation). In unstimulated cells, ERK-mediated negative feedback reduced the population-average and cell-cell variability of the level of activated ppERK and increased its robustness to changes in ERK expression. In stimulated cells, negative feedback (evident between 5 min and 4 h) also reduced average levels and variability of phosphorylated ERK (ppERK) without altering the "gradedness" or sensitivity of the response. Binning cells according to total ERK expression revealed, strikingly, that maximal ppERK responses initially occur at submaximal ERK levels and that this non-monotonic relationship changes to an increasing, monotonic one within 15 min. These phenomena occur in HeLa cells and MCF7 breast cancer cells and in the presence and absence of ERK-mediated negative feedback. They were best modeled assuming distributive (rather than processive) activation. Thus, we have uncovered a novel, time-dependent change in the relationship between total ERK and ppERK levels that persists without negative feedback. This change makes acute response kinetics dependent on ERK level and provides a "gating" or control mechanism in which the interplay between stimulus duration and the distribution of ERK expression across cells could modulate the proportion of cells that respond to stimulation.

Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis.

Glycated proteins are important biomarkers for age-related disorders, however their analysis is challenging because of the complexity of the protein-carbohydrate adducts. Here we report a method that enables the detection and identification of individual glycated proteins in complex samples using fluorescent boronic acids in gel electrophoresis. Using this method we identified glycated proteins in human serum, insect hemolymph and mouse brain homogenates, confirming this technique as a powerful proteomics tool that can be used for the identification of potential disease biomarkers.

Dual-specificity MAP kinase phosphatases (MKPs): shaping the outcome of MAP kinase signalling.

Dual-specificity MAP kinase phosphatases (MKPs) provide a complex negative regulatory network that acts to shape the duration, magnitude and spatiotemporal profile of MAP kinase activities in response to both physiological and pathological stimuli. Individual MKPs may exhibit either exquisite specificity towards a single mitogen-activated protein kinase (MAPK) isoform or be able to regulate multiple MAPK pathways in a single cell or tissue. They can act as negative feedback regulators of MAPK activity, but can also provide mechanisms of crosstalk between distinct MAPK pathways and between MAPK signalling and other intracellular signalling modules. In this review, we explore the current state of knowledge with respect to the regulation of MKP expression levels and activities, the mechanisms by which individual MKPs recognize and interact with different MAPK isoforms and their role in the spatiotemporal regulation of MAPK signalling.

Mechanisms of GnRH-induced extracellular signal-regulated kinase nuclear localization.

Gonadotropin-releasing hormone receptors (GnRHR) mediate activation and nuclear translocation of the extracellular signal regulated kinases 1 and 2 (ERK) by phosphorylation on the TEY motif. This is necessary for GnRH to initiate transcriptional programmes controlling fertility, but mechanisms that govern ERK targeting are unclear. Using automated microscopy to explore ERK regulation in single cells, we find that GnRHR activation induces marked redistribution of ERK to the nucleus and that this effect can be uncoupled from the level of TEY phosphorylation of ERK. Thus, 5 min stimulation with 100 nM GnRH increased phospho-ERK levels (from 89 ± 34 to 555 ± 45 arbitrary fluorescence units) and increased the nuclear:cytoplasmic (N:C) ERK ratio (from 1.36 ± 0.06 to 2.16 ± 0.05) in the whole cell population, but it also significantly increased N:C ERK in cells binned according to phospho-ERK levels. This phosphorylation unattributable component of the ERK translocation response occurs at a broad range of GnRHR expression levels, in the presence of tyrosine phosphatase and protein synthesis inhibitors, and in ERK mutants unable to undergo catalytic activation. It also occurred in mutants incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK binding partners. It was however, reduced by MEK or PKC inhibition and by mutations preventing TEY phosphorylation or that abrogate ERK binding to D (docking) domain partners. We therefore show that TEY phosphorylation of ERK is necessary, but not sufficient for the full nuclear localization response. We further show that this "phosphorylation unattributable" component of GnRH-mediated ERK nuclear translocation requires both PKC activity and association with partner proteins via the D-domain.

ERK phosphorylation and nuclear accumulation: insights from single-cell imaging.

Many stimuli mediate activation and nuclear translocation of ERK (extracellular-signal-regulated kinase) by phosphorylation on the TEY (Thr-Glu-Tyr) motif. This is necessary to initiate transcriptional programmes controlling cellular responses, but the mechanisms that govern ERK nuclear targeting are unclear. Single-cell imaging approaches have done much to increase our understanding of input-output relationships in the ERK cascade, but few studies have addressed how the range of ERK phosphorylation responses observed in cell populations influences subcellular localization. Using automated microscopy to explore ERK regulation in single adherent cells, we find that nuclear localization responses increase in proportion to stimulus level, but not the level of TEY phosphorylation. This phosphorylation-unattributable nuclear localization response occurs in the presence of tyrosine phosphatase and protein synthesis inhibitors. It is also seen with a catalytically inactive ERK2-GFP (green fluorescent protein) mutant, and with a mutant incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK-binding partners. It is, however, reduced by MEK (mitogen-activated protein kinase/ERK kinase) inhibition and by mutations preventing TEY phosphorylation or in the ERK common docking region. We therefore show that TEY phosphorylation of ERK is necessary, but not sufficient, for the full nuclear accumulation response and that this 'phosphorylation-unattributable' component of stimulus-mediated ERK nuclear localization requires association with partner proteins via the common docking motif.

Decoding neurohormone pulse frequency by convergent signalling modules.

GnRH (gonadotropin-releasing hormone) mediates control of reproduction. It is secreted in pulses and acts via intracellular effectors to activate gene expression. Submaximal GnRH pulse frequency can elicit maximal responses, yielding bell-shaped frequency-response curves characteristic of genuine frequency decoders. GnRH frequency decoding is therapeutically important (pulsatile GnRH can drive ovulation in assisted reproduction, whereas sustained activation can treat breast and prostate cancers), but the mechanisms are unknown. In the present paper, we review recent work in this area, placing emphasis on the regulation of transcription, and showing how mathematical modelling of GnRH effects on two effectors [ERK (extracellular-signal-regulated kinase) and NFAT (nuclear factor of activated T-cells)] reveals the potential for genuine frequency decoding as an emergent feature of the GnRH signalling network, rather than an intrinsic feature of a given protein or pathway within it.

Dual specificity phosphatases 10 and 16 are positive regulators of EGF-stimulated ERK activity: indirect regulation of ERK signals by JNK/p38 selective MAPK phosphatases.

We have explored the possible role of dual specificity phosphatases (DUSPs) on acute EGF-mediated ERK signalling using high content imaging and a delayed MEK inhibition protocol to distinguish direct and indirect effects of the phosphatases on ERK activity. Using siRNAs, we were unable to find evidence that any of the MAPK phosphatases (MKPs) expressed in HeLa cells acts directly to dephosphorylate ppERK1/2 (dual phosphorylated ERKs 1 and/or 2) in the acute time-frame tested (0-14 min). Nevertheless, siRNAs against two p38/JNK MKPs (DUSPs 10 and 16) inhibited acute EGF-stimulated ERK activation. No such effect was seen for acute effects of the protein kinase C activator PDBu (phorbol 12,13 dibutyrate) on ERK activity, although effects of EGF and PDBu on ERK-dependent transcription (Egr-1 luciferase activity) were both reduced by siRNA targeting DUSPs 10 and 16. Inhibition of EGF-stimulated ERK activity by these siRNAs was reversed by pharmacological inhibition of p38 MAPK and single cell analysis revealed that the siRNAs did not influence the nuclear-cytoplasmic distribution of ppERK1/2. Thus, DUSPs 10 and 16 are positive regulators of activation, apparently acting by modulating cross-talk between the p38 and ERK pathways. A simplified mathematical model of this scenario accurately predicted the experimental data, supporting the conclusion that the major mechanism by which MKPs influence acute EGF-stimulated ERK responses is the negative regulation of p38, resulting in the positive regulation of ERK phosphorylation and activity.

Decoding GnRH neurohormone pulse frequency by convergent signalling modules.

Gonadotropin-releasing hormone (GnRH) mediates control of reproduction. It is secreted in pulses and acts via intracellular effectors to activate gonadotrophin secretion and gene expression. Sub-maximal GnRH pulse frequency can elicit maximal responses, yielding bell-shaped frequency-response curves characteristic of genuine frequency decoders. GnRH frequency decoding is therapeutically important (pulsatile GnRH can drive ovulation in assisted reproduction whereas sustained activation can treat breast and prostate cancers), but the mechanisms are unknown. Here, we consider the possibility that it is due to convergence of distinct pulsatile signals at the transcriptome. We develop a model that mirrors wet-laboratory data for activation and nuclear translocation of GnRH effectors (extracellular signal regulated kinase and nuclear factors of activated T-cells) and incorporates transcription. The model predicts genuine frequency decoding when two transcription factors (TFs) converge at a cooperative gate, and shows how optimal pulse frequency could reflect TF activation kinetics and affinities. Importantly, this behaviour is revealed as an emergent feature of the network, rather than an intrinsic feature of a given protein or pathway, and since such network topology is extremely common, may well be widespread in biological systems.

Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells.

Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors.