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The demand for foreign teachers of English as a Foreign Language (EFL) has been high in non-English speaking countries, including Thailand. As foreigners have resided in the country for several years, they picked up students’ first language (L1) competence. Several foreign teachers, to some extent, utilized this mastery of L1 to assist the English instruction. This research aimed to explore how students with different Common European Framework of Reference for Languages (CEFR) levels perceive foreign English teachers’ use of L1. Three hundred twenty students from Walailak University participated in the survey. A quantitative survey and qualitative interview were used to collect data. Descriptive and inferential statistics were performed to analyze quantitative data, while content analysis was administered to investigate the qualitative data. The quantitative findings revealed a significant difference in students’ perceptions, Welch’s F(2,68.42)=11.304, p .05. The qualitative findings exposed that students in level A1 had significantly different perceptions compared to those in levels A2 and B1. Students who supported L1 integration disclosed that it ameliorated communication, enhanced learning motivation, and improved academic achievement. Additionally, students who opposed L1 expressed that English’s full usage developed their learning motivation and academic accomplishment.

The problem of ensuring the sustainability of the processing process is directly related to the energy efficiency of the entire processing process. Virtually any machine the control of motion of which is considered interacts with the environment (technological, aerodynamic, trybologic, etc.). If one sticks to the synergetic analytical concept, the influence of the environment is described by a model of forces in state coordinates. The interaction is also characterized by the irreversible transformation capacity trajectory on the work. As a result, the system parameters are change causing additional strain displacements changing the trajectory. This transformation characterizes internal control and they are defined not by external action but by internal self-organization and defines the evolutionary trajectory. The properties of the system along the evolutionary trajectory change and the trajectory can lose its stability. The article considers the process of mathematical simulation of the evolutionary trajectory forming by the example of metalwork by cutting at turning machines and analyzes its stability. The evolution is presented as the parameters of the system depending on the capacity and work trajectory and presented by the Volterra integral equation of the second kind. When the trajectory loses its stability, irreversible changes can be formed in the system which may cause a complete system breakdown. In addition, the loss of stability leads to an increase in the oscillation amplitude of the tool and, as a consequence, an increase in energy costs for processing.

The problem of ensuring stability of movements of the tool about workpiece taking into account parametric self-excitation is considered. The factors causing periodic changes of parameters in dynamic system when cutting are analyzed. They are connected with spatial anisotropy of properties of elasticity in a subsystem of the processed workpiece with allowance variations on workpiece rotation perimeter, with kinematic perturbation from mechanical part of drives of executive elements of the machine, with periodic processes in a cutting zone. The generalized dynamic model of system with periodically changing parameters is given. Influence of periodically changing parameters on stability of trajectories is studied. The features of loss of stability specific to dynamic systems of cutting are disclosed. In particular, it is shown that due to parametrical effects with increasing cutting speed at increase of rotational frequency of spindle there is always a critical frequency at which the system loses stability.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, Myc P59A , a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P +1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors., Competing Interests: JA, DV, DJ, LP, NA No competing interests declared, GV Guy Vigers is Structural Biology Consultant at Allium Consulting LLC, HC Dr. Huifen Chen is Distinguished Scientist at Genentech, Inc, JM Dr. John Moffat is Distinguished Scientist at Genentech, Inc, (© 2023, Anderson et al.)

The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile ("3P"), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although copolarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.

Patients with melanoma receiving drugs targeting BRAFV600E and mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2) invariably develop resistance and face continued progression. Based on preclinical studies, intermittent treatment involving alternating periods of drug withdrawal and rechallenge has been proposed as a method to delay the onset of resistance. The beneficial effect of intermittent treatment has been attributed to drug addiction, where drug withdrawal reduces the viability of resistant cells due to MAP kinase pathway hyperactivation. However, the mechanistic basis of the intermittent effect is incompletely understood. We show that intermittent treatment with the BRAFV600E inhibitor, LGX818/encorafenib, suppresses growth compared with continuous treatment in human melanoma cells engineered to express BRAFV600E, p61-BRAFV600E, or MEK2C125 oncogenes. Analysis of the BRAFV600E-overexpressing cells shows that, while drug addiction clearly occurs, it fails to account for the advantageous effect of intermittent treatment. Instead, growth suppression is best explained by resensitization during periods of drug removal, followed by cell death after drug readdition. Continuous treatment leads to transcriptional responses prominently associated with chemoresistance in melanoma. By contrast, cells treated intermittently reveal a subset of transcripts that reverse expression between successive cycles of drug removal and rechallenge and include mediators of cell invasiveness and the epithelial-to-mesenchymal transition. These transcripts change during periods of drug removal by adaptive switching, rather than selection pressure. Resensitization occurs against a background of sustained expression of melanoma resistance genes, producing a transcriptome distinct from that of the initial drug-naive cell state. We conclude that phenotypic plasticity leading to drug resensitization can underlie the beneficial effect of intermittent treatment.

Sexual self-concept (SSC) is an important component of health that is closely associated with the well-being of survivors of child sexual abuse (CSA). Compared with non-CSA respondents, SSC of CSA survivors was deleteriously affected, resulting in ineffective psychological functioning (negative self-evaluation, psychological ill-health, and difficulties in interpersonal relationships). Negative SSC (sexual anxiety, sexual depression, and sexual fear) played a significant role in mediating the adverse effects of CSA on psychological functioning. Future studies may recruit a sample with greater variation in CSA characteristics and use externalizing problems as outcome measures to cross-validate findings of this study., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 Taylor & Francis Group, LLC.)

Human catechol O -methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S -adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor-acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954-7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15-20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate., Competing Interests: The authors declare no competing interest.

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors., Competing Interests: Conflict of interest statement: G.P.V., D.S., and H.Z. are employees of Array BioPharma Inc.; W.W. and J.G.M. are employees of Genentech Inc.; and H.Z. is the CEO of Blueray Biopharma.

The BRAF-MKK1/2-ERK1/2 pathway is constitutively activated in response to oncogenic mutations of BRAF in many cancer types, including melanoma. Although small molecules that inhibit oncogenic BRAF and MAP kinase kinase (MKK)1/2 have been successful in clinical settings, resistance invariably develops. High affinity inhibitors of ERK1/2 have been shown in preclinical studies to bypass the resistance of melanoma and colon cancer cells to BRAF and MKK1/2 inhibitors, and are thus promising additions to current treatment protocols. But still unknown is how molecular responses to ERK1/2 inhibitors compare with inhibitors currently in clinical use. Here, we employ quantitative phosphoproteomics to evaluate changes in phosphorylation in response to the ERK inhibitors, SCH772984 and GDC0994, and compare these to the clinically used MKK1/2 inhibitor, trametinib. Combined with previous studies measuring phosphoproteomic responses to the MKK1/2 inhibitor, selumetinib, and the BRAF inhibitor, vemurafenib, the outcomes reveal key insights into pathway organization, phosphorylation specificity and off-target effects of these inhibitors. The results demonstrate linearity in signaling from BRAF to MKK1/2 and from MKK1/2 to ERK1/2. They identify likely targets of direct phosphorylation by ERK1/2, as well as inhibitor off-targets, including an off-target regulation of the p38α mitogen activated protein kinase (MAPK) pathway by the MKK1/2 inhibitor, trametinib, at concentrations used in the literature but higher than in vivo drug concentrations. In addition, several known phosphorylation targets of ERK1/2 are insensitive to MKK or ERK inhibitors, revealing variability in canonical pathway responses between different cell systems. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gamma irradiation is able to affect various structural modification and an increase of the biological properties of biomaterials. This study was conducted to investigate the anti-allergenic effect of γ-irradiated black ginseng extract (BGE) using in vitro and in vivo experiments. IgEantigen complex-induced degranulation was measured in RBL-2H3 mast cells. In addition, an anti-atopic dermatitis (AD) test was carried out by spreading γ-irradiated BGE on the dorsal skin of 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice. The content of arginylfructose (AF) of gamma-irradiated BGE was higher than that of BGE. In RBL-2H3 mast cells, γ-irradiated BGE treatments significantly reduced the IgE-antigen complex-induced release of β-hexosaminidase, histamine, intracellular ROS, and Ca2+ influx. A western blot analysis showed that γ-irradiated BGE had an inhibitory activity on the FcεRI-mediated signaling in mast cells. In the DNCB-induced AD model, γ-irradiated BGE significantly alleviated the ADlike skin symptoms and clinical signs. The suppression of AD by γ-irradiated BGE was accompanied by a decrease in the serum level of IgE and IL-4, as well as the number of leukocyte. Gamma-irradiated BGE also suppressed IL-4 and increased IFN-γ in splenocytes. Our data suggests that γ-irradiated BGE may be effective therapeutic agents for the treatment of AD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration., (© 2017 Connacher et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Background: The chemical constituents of Panax ginseng are changed by processing methods such as steaming or sun drying. In the present study, the chemical change of Panax ginseng induced by steaming was monitored in situ ., Methods: Samples were separated from the same ginseng root by incision during the steaming process, for in situ monitoring. Sampling was sequentially performed in three stages; FG (fresh ginseng) → SG (steamed ginseng) → RG (red ginseng) and 60 samples were prepared and freeze dried. The samples were then analyzed to determine 43 constituents among three stages of P. ginseng ., Results: The results showed that six malonyl-ginsenoside (Rg1, Rb1, Rb3, Rc, Rd, Rb2) and 15 amino acids were decreased in concentration during the steaming process. In contrast, ginsenoside-Rh1, 20( S )-Rg2, 20( S, R )-Rg3 and Maillard reaction product such as AF (arginine-fructose), AFG (arginine-fructose-glucose), and maltol were newly generated or their concentrations were increased., Conclusion: This study elucidates the dynamic changes in the chemical components of P. ginseng when the steaming process was induced. These results are thought to be helpful for quality control and standardization of herbal drugs using P. ginseng and they also provide a scientific basis for pharmacological research of processed ginseng (Red ginseng).

Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4) 2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4) 2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4) 2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4) 2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication., Competing Interests: JKT: Senior editor, eLife. The other authors declare that no competing interests exist.

MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect., (© 2016 Goshen-Lago, Goldberg-Carp, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Many enzymes are self-regulated and can either inhibit or enhance their own catalytic activity. Enzymes that do both are extremely rare. Many protein kinases autoactivate by autophosphorylating specific sites at their activation loop and are inactivated by phosphatases. Although mitogen-activated protein kinases (MAPKs) are usually activated by dual phosphorylation catalyzed by MAPK kinases (MAPKKs), the MAPK p38β is exceptional and is capable of self-activation by cis autophosphorylation of its activation loop residue T180. We discovered that p38β also autophosphorylates in trans two previously unknown sites residing within a MAPK-specific structural element known as the MAPK insert: T241 and S261. Whereas phosphorylation of T180 evokes catalytic activity, phosphorylation of S261 reduces the activity of T180-phosphorylated p38β, and phosphorylation of T241 reduces its autophosphorylation in trans Both phosphorylations do not affect the activity of dually phosphorylated p38β. T241 of p38β is found phosphorylated in vivo in bone and muscle tissues. In myogenic cell lines, phosphorylation of p38β residue T241 is correlated with differentiation to myotubes. T241 and S261 are also autophosphorylated in intrinsically active variants of p38α, but in this protein, they probably play a different role. We conclude that p38β is an unusual enzyme that automodulates its basal, MAPKK-independent activity by several autophosphorylation events, which enhance and suppress its catalytic activity., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk's activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation., (© 2016 Smorodinsky-Atias, Goshen-Lago, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Here, we demonstrate the flexibility of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels for modeling tumor progression. The PEG hydrogels were formed using thiol-ene chemistry to incorporate a matrix metalloproteinase-degradable peptide crosslinker (KKCGGPQG↓IWGQGCKK) permissive to proteolytic remodeling and the adhesive CRGDS peptide ligand. Tumor cell function was investigated by culturing WM239A melanoma cells on PEG hydrogel surfaces or encapsulating cells within the hydrogels, and either as monocultures or indirect (non-contact) cocultures with primary human dermal fibroblasts (hDFs). WM239A cluster size and proliferation rate depended on the shear elastic modulus for cells cultured on PEG hydrogels, while growth was inhibited by coculture with hDFs regardless of hydrogel stiffness. Cluster size was also suppressed by hDFs for WM239A cells encapsulated in PEG hydrogels, which is consistent with cells seeded on top of hydrogels. Notably, encapsulated WM239A clusters and single cells adopted invasive phenotypes in the hDF coculture model, which included single cell and collective migration modes that resembled invasion from human melanoma patient-derived xenograft tumors encapsulated in equivalent PEG hydrogels. Our combined results demonstrate that peptide-functionalized PEG hydrogels provide a useful platform for investigating aspects of tumor progression in 2D and 3D microenvironments, including single cell migration, cluster growth and invasion.

It has been reported that Korean Red Ginseng has been manufactured for 1,123 y as described in the GoRyeoDoGyeong record. The Korean Red Ginseng manufactured by the traditional preparation method has its own chemical component characteristics. The ginsenoside content of the red ginseng is shown as Rg1: 3.3 mg/g, Re: 2.0 mg/g, Rb1: 5.8 mg/g, Rc:1.7 mg/g, Rb2: 2.3 mg/g, and Rd: 0.4 mg/g, respectively. It is known that Korean ginseng generally consists of the main root and the lateral or fine roots at a ratio of about 75:25. Therefore, the red ginseng extract is prepared by using this same ratio of the main root and lateral or fine roots and processed by the historical traditional medicine prescription. The red ginseng extract is prepared through a water extraction (90(°)C for 14-16 h) and concentration process (until its final concentration is 70-73 Brix at 50-60(°)C). The ginsenoside contents of the red ginseng extract are shown as Rg1: 1.3 mg/g, Re: 1.3 mg/g, Rb1: 6.4 mg/g, Rc:2.5 mg/g, Rb2: 2.3 mg/g, and Rd: 0.9 mg/g, respectively. Arginine-fructose-glucose (AFG) is a specific amino-sugar that can be produced by chemical reaction of the process when the fresh ginseng is converted to red ginseng. The content of AFG is 1.0-1.5% in red ginseng. Acidic polysaccharide, which has been known as an immune activator, is at levels of 4.5-7.5% in red ginseng. Therefore, we recommended that the chemical profiles of Korean Red Ginseng made through the defined traditional method should be well preserved and it has had its own chemical characteristics since its traditional development.

Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hedgehog (HH) proteins are proteolytically processed into a biologically active form that is covalently modified by cholesterol and palmitate. However, most studies of HH biogenesis have characterized protein from cells in which HH is overexpressed. We purified Sonic Hedgehog (SHH) from cells expressing physiologically relevant levels and showed that it was more potent than SHH isolated from overexpressing cells. Furthermore, the SHH in our preparations was modified with a diverse spectrum of fatty acids on its amino termini, and this spectrum of fatty acids varied dramatically depending on the growth conditions of the cells. The fatty acid composition of SHH affected its trafficking to lipid rafts as well as its potency. Our results suggest that HH proteins exist as a family of diverse lipid-speciated proteins that might be altered in different physiological and pathological contexts in order to regulate distinct properties of HH proteins., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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 p38β, 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 p38β. 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 p38β in mammalian cells. Thus, this study identified the structural motif responsible for the unique autophosphorylation capability of p38β 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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR (13)C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with kex ≈ 300 s(-1) (kAB ≈ 240 s(-1)/kBA ≈ 60 s(-1)) and pA/pB ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of kex ≈ 500 s(-1) (kAB ≈ 15 s(-1)/kBA ≈ 485 s(-1)) and pA/pB ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.

Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT-folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 bound the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels ("synthetic extracellular matrix" or "synthetic ECM"). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

Wnt5a directs the assembly of the Wnt-receptor-actin-myosin-polarity (WRAMP) structure, which integrates cell-adhesion receptors with F-actin and myosin to form a microfilament array associated with multivesicular bodies (MVBs). The WRAMP structure is polarized to the cell posterior, where it directs tail-end membrane retraction, driving forward translocation of the cell body. Here we define constituents of the WRAMP proteome, including regulators of microfilament and microtubule dynamics, protein interactions, and enzymatic activity. IQGAP1, a scaffold for F-actin nucleation and crosslinking, is necessary for WRAMP structure formation, potentially bridging microfilaments and MVBs. Vesicle coat proteins, including coatomer-I subunits, localize to and are required for the WRAMP structure. Electron microscopy and live imaging demonstrate movement of the ER to the WRAMP structure and plasma membrane, followed by elevation of intracellular Ca2+. Thus, Wnt5a controls directional movement by recruiting cortical ER to mobilize a rear-directed, localized Ca2+ signal, activating actomyosin contraction and adhesion disassembly for membrane retraction., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Over two-thirds of melanomas have activating mutations in B-Raf, leading to constitutive activation of the B-Raf/MKK/ERK signaling pathway. The most prevalent mutation, B-RafV600E, promotes cancer cell behavior through mechanisms that are still incompletely defined. Here, we used a sensitive microarray profiling platform to compare microRNA (miRNA) expression levels between primary melanocytes and B-RafV600E-positive melanoma cell lines, and between melanoma cells treated in the presence and absence of an MKK1/2 inhibitor. We identified a network of >20 miRNAs deregulated by B-Raf/MKK/ERK in melanoma cells, the majority of which modulate the expression of key cancer regulatory genes and functions. Importantly, miRNAs within the network converge on protein regulation and cancer phenotypes, suggesting that these miRNAs might function combinatorially. We show that miRNAs augment effects on protein repression and cell invasion when co-expressed, and gene-specific latency and interference effects between miRNAs were also observed. Thus, B-Raf/MKK/ERK controls key aspects of cancer cell behavior and gene expression by modulating a network of miRNAs with cross-regulatory functions. The findings highlight the potential for complex interactions between coordinately regulated miRNAs within a network.

Side population (SP) cells are identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and it represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a substrate of ABCB1, both in vitro and in vivo. However, melanoma SP cells were also resistant to temozolomide, which is not a substrate for ABC transporters, through IL-8 upregulation. In addition, gene profiling studies identified three signaling pathways (NF-κB, α6-β4-integrin, and IL-1) as differentially upregulated in melanoma SP cells, and there was a significant increase of PCDHB11 and decrease of FUK and TBX2 in these cells. Therefore, we provide evidence that SP is an enriched source of chemoresistant cells in human melanomas, and suggest that the selected genes and signaling pathways of SP cells may be a potential target for effective melanoma therapies. To our knowledge, this is a previously unreported study to isolate SP cells from melanoma patients and to investigate the gene expression profiling of these cells.

For quality control of components in Korean red ginseng powder and extract, a new method for simultaneous quantification of 12 ginsenosides (Rg1, Re, Rf, Rh1, Rg2[S], Rg2[R], Rb1, Rc, Rb2, Rd, Rg3[S], and Rg3[R]) was studied. Compared to the official method for quantification of marker substances (ginsenosides Rg1 and Rb1), the proposed methods were guaranteed by in-house method validation. Several criteria such as linearity, specificity, precision and accuracy were evaluated. For red ginseng powder, recovery (averaging 95% to 105%) was calculated, and analysis of variance was carried out to estimate the relative standard deviation (0.20% to 2.12%). For red ginseng extract, the average recovery rate was 90% to 99% and the relative standard deviation was 0.39% to 2.40%. These results indicate that the proposed method could be used in the laboratory for determination of 12 ginsenosides in red ginseng powder and extract. In addition, this method was found to be suitable for quality control of ginseng products and potentially offer time and cost benefits.

Argonaute proteins (Ago1-4) are essential components of the microRNA-induced silencing complex and play important roles in both microRNA biogenesis and function. Although Ago2 is the only one with the slicer activity, it is not clear whether the slicer activity is a universally critical determinant for Ago2's function in mammals. Furthermore, functional specificities associated with different Argonautes remain elusive. Here we report that microRNAs are randomly sorted to individual Argonautes in mammals, independent of the slicer activity. When both Ago1 and Ago2, but not either Ago1 or Ago2 alone, are ablated in the skin, the global expression of microRNAs is significantly compromised and it causes severe defects in skin morphogenesis. Surprisingly, Ago3 is able to load microRNAs efficiently in the absence of Ago1 and Ago2, despite a significant loss of global microRNA expression. Quantitative analyses reveal that Ago2 interacts with a majority of microRNAs (60%) in the skin, compared with Ago1 (30%) and Ago3 (<10%). This distribution is highly correlated with the abundance of each Argonaute, as quantified by shotgun proteomics. The quantitative correlation between Argonautes and their associated microRNAs is conserved in human cells. Finally, we measure the absolute expression of Argonaute proteins and determine that their copy number is ~1.4 × 10(5) to 1.7 × 10(5) molecules per cell. Together, our results reveal a quantitative picture for microRNA activity in mammals.

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies.

Hydrogen/deuterium exchange measurements by mass spectrometry (HX-MS) can be used to report localized conformational mobility within folded proteins, where exchange predominantly occurs through low energy fluctuations in structure, allowing transient solvent exposure. Changes in conformational mobility may impact protein function, even in cases where structural changes are unobservable. Previous studies of the MAP kinase, ERK2, revealed increases in HX upon activation occured at the hinge between conserved N- and C-terminal domains, which could be ascribed to enhanced backbone flexibility. This implied that kinase activation modulates interdomain closure, and was supported by evidence for two modes of nucleotide binding that were consistent with closed vs open conformations in active vs inactive forms of ERK2, respectively. Thus, phosphorylation of ERK2 releases constraints to interdomain closure, by modulating hinge flexibility. In this study, we examined ERK1, which shares 90% sequence identity with ERK2. HX-MS measurements of ERK1 showed similarities with ERK2 in overall deuteration, consistent with their similar tertiary structures. However, the patterns of HX that were altered upon activation of ERK1 differed from those in ERK2. In particular, alterations in HX at the hinge region upon activation of ERK2 did not occur in ERK1, suggesting that the two enzymes differ with respect to their regulation of hinge mobility and interdomain closure. In agreement, HX-MS measurements of nucleotide binding suggested revealed domain closure in both inactive and active forms of ERK1. We conclude that although ERK1 and ERK2 are closely related with respect to primary sequence and tertiary structure, they utilize distinct mechanisms for controlling enzyme function through interdomain interactions.

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.

In this issue of Molecular Cell, Hoshi et al. (2010) report two examples in which small molecule inhibitors are rendered ineffective when their kinase targets are involved in protein-protein interactions, highlighting differences between in vivo and in vitro inhibition kinetics., (Copyright 2010 Elsevier Inc. All rights reserved.)

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Human melanomas show oncogenic B-Raf mutations, which activate the B-Raf/MKK/ERK cascade. We screened microarrays to identify cellular targets of this pathway, and found that genes upregulated by B-Raf/MKK/ERK showed highest association with cell-cycle regulators, whereas genes downregulated were most highly associated with axon guidance genes, including plexin-semaphorin family members. Plexin B1 was strongly inhibited by mitogen-activated protein kinase signaling in melanoma cells and melanocytes. In primary melanoma cells, plexin B1 blocked tumorigenesis as measured by growth of colonies in soft agar, spheroids in extracellular matrix and xenograft tumors. Tumor suppression depended on residues in the C-terminal domain of plexin B1, which mediate receptor GTPase activating protein activity, and also correlated with AKT inhibition. Interestingly, the inhibitory response to plexin B1 was reduced or absent in cells from a matched metastatic tumor, suggesting that changes occur in metastatic cells which bypass the tumor-suppressor mechanisms. Plexin B1 also inhibited cell migration, but this was seen in metastatic cells and not in matched primary cells. Thus, plexin B1 has tumor-suppressor function in early-stage cells, although suppressing migration in late-stage cells. Our findings suggest that B-Raf/MKK/ERK provides a permissive environment for melanoma genesis by modulating plexin B1.

Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.

MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2(R65S+D319N) and Rolled(R80S+D334N)) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2(R65S) and Erk2(R65S+D319N) were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained.

Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.

The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr(675), Thr(676), and Thr(686), but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr(676). Mutation of Thr(676) in hMps1 or the corresponding Thr(591) residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo.

A major limitation in identifying peptides from complex mixtures by shotgun proteomics is the ability of search programs to accurately assign peptide sequences using mass spectrometric fragmentation spectra (MS/MS spectra). Manual analysis is used to assess borderline identifications; however, it is error-prone and time-consuming, and criteria for acceptance or rejection are not well defined. Here we report a Manual Analysis Emulator (MAE) program that evaluates results from search programs by implementing two commonly used criteria: 1) consistency of fragment ion intensities with predicted gas phase chemistry and 2) whether a high proportion of the ion intensity (proportion of ion current (PIC)) in the MS/MS spectra can be derived from the peptide sequence. To evaluate chemical plausibility, MAE utilizes similarity (Sim) scoring against theoretical spectra simulated by MassAnalyzer software (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922) using known gas phase chemical mechanisms. The results show that Sim scores provide significantly greater discrimination between correct and incorrect search results than achieved by Sequest XCorr scoring or Mascot Mowse scoring, allowing reliable automated validation of borderline cases. To evaluate PIC, MAE simplifies the DTA text files summarizing the MS/MS spectra and applies heuristic rules to classify the fragment ions. MAE output also provides data mining functions, which are illustrated by using PIC to identify spectral chimeras, where two or more peptide ions were sequenced together, as well as cases where fragmentation chemistry is not well predicted.

Studies of protein kinases have identified a "gatekeeper" residue, which confers selectivity for binding nucleotides and small-molecule inhibitors. We report that, in the MAP kinase ERK2, mutations at the gatekeeper residue unexpectedly lead to autoactivation due to enhanced autophosphorylation of regulatory Tyr and Thr sites within the activation lip that control kinase activity. This occurs through an intramolecular mechanism, indicating that the gatekeeper residue indirectly constrains flexibility at the activation lip, precluding access of the phosphoacceptor residues to the catalytic base. Other residues that interact with the gatekeeper site to form a hydrophobic cluster in the N-terminal domain also cause autoactivation when mutated. Hydrogen-exchange studies of a mutant within this cluster reveal perturbations in the conserved DFG motif, predicting a route for side chain connectivity from the hydrophobic cluster to the activation lip. Mutations of residues along this route support this model, explaining how information about the gatekeeper residue identity can be transmitted to the activation lip. Thus, an N-terminal hydrophobic cluster that includes the gatekeeper forms a novel structural unit, which functions to maintain the "off" state of ERK2 before cell signal activation.