Equilibrium dissociation constants for RAS-effector interaction determined Fluorescence polarization.

<p>(A) Fluorescence polarization experiments were conducted by titrating mGppNHp-bound, active forms of RAS proteins (1 μM, respectively) with increasing concentrations of the respective effector domains as MBP fusion proteins. Data of two representative experiments for the interaction of KRAS (upper panel) and RRAS2 (lower panel) with CRAF-RB and PI3Kα-RB, respectively, are shown. All other data are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167145#pone.0167145.s002" target="_blank">S1 Fig</a> (B) Evaluated equilibrium dissociation constants (K_d) in μM shown as data points illustrate a significant difference in the binding properties of the effector proteins with both RAS and RRAS isoforms, respectively. A mean value of 0.94 ± 0.014 μM has been determined for the interaction between HRAS and CRAF to exemplify the reproducibility of this approach.</p

The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

<div><p>RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our <i>in vitro</i> data with <i>in silico</i> data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS <i>versus</i> RRAS isoforms with effectors.</p></div

Register of dissociation constants (K_d) determined for the RAS-effector interactions.

<p>Register of dissociation constants (K_d) determined for the RAS-effector interactions.</p

Domain organization of RAS effectors and different proteins used in this study.

<p>(A) Various domains are highlighted, including RAS association domain (RA) and RAS-binding (RB) domain in blue. The numbers indicate the N- and C-terminal amino acids of the respective effector domain used in this study. Other domains are: C1, cysteine-rich lipid binding; C2, calcium-dependent lipid binding; CRD, cysteine rich domains; DEP, Dishevelled/Egl-10/Pleckstrin; EF, EF-hand; kinase, serine/threonine or phosphoinositide kinase; PH, pleckstrin homology; PI3K, Phosphoinositide 3-kinase family, accessory <i>domain;</i> PP, proline-rich region; RA, RAS association; RALGEF, RAL specific guanine nucleotide exchange factor; RASGEF, RAS specific guanine nucleotide exchange factor; RB, RAS binding; REM, RAS exchanger motif; SARAH, Salvador/RASSF/Hippo. (B) Coomassie brilliant blue (CBB) stained SDS-PAGE of purified MBP fusion proteins used in this study.</p

RAS-effector interaction hotspots.

<p>(A) Interaction matrix of RAS isoforms and effector proteins. Interaction matrix is launched to demonstrate interaction residues in all available structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167145#pone.0167145.g003" target="_blank">Fig 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167145#pone.0167145.s005" target="_blank">S4 Fig</a>). Left and upper parts comprise the amino acid sequence alignments of the RAS proteins and the effector domains, respectively. Each element corresponds to a possible interaction of RAS (row; HRAS numbering) and effector (column; CRAF numbering) residues. As indicated, interaction matrix represents five main regions, which cover the main interacting interfaces. (B) The five main regions, comprising the main hotspot for the RAS-effector interaction, are highlighted as ribbon and surface representations in the corresponding colors for the structures of HRAS-PLCε (PDB code: 2C5L) and HRAS-CRAF (PDB code: 4G0N). Key amino acids which are highlighted by colored background in A are indicated on the structures as well.</p

DataSheet1_New mechanistic insights into the RAS-SIN1 interaction at the membrane.pdf

Stress-activated MAP kinase-interacting protein 1 (SIN1) is a central member of the mTORC2 complex that contains an N-terminal domain (NTD), a conserved region in the middle (CRIM), a RAS-binding domain (RBD), and a pleckstrin homology domain. Recent studies provided valuable structural and functional insights into the interactions of SIN1 and the RAS-binding domain of RAS proteins. However, the mechanism for a reciprocal interaction of the RBD-PH tandem with RAS proteins and the membrane as an upstream event to spatiotemporal mTORC2 regulation is not clear. The biochemical assays in this study led to the following results: 1) all classical RAS paralogs, including HRAS, KRAS4A, KRAS4B, and NRAS, can bind to SIN1-RBD in biophysical and SIN1 full length (FL) in cell biology experiments; 2) the SIN1-PH domain modulates interactions with various types of membrane phosphoinositides and constantly maintains a pool of SIN1 at the membrane; and 3) a KRAS4A-dependent decrease in membrane binding of the SIN1-RBD-PH tandem was observed, suggesting for the first time a mechanistic influence of KRAS4A on SIN1 membrane association. Our study strengthens the current mechanistic understanding of SIN1-RAS interaction and suggests membrane interaction as a key event in the control of mTORC2-dependent and mTORC2-independent SIN1 function.</p

Activity-induced increase of pERK is abolished in Ptpn11^D61Y neurons and can be restored upon MEK1 inhibition.

<p>Staining <b>(A)</b> and quantification <b>(B)</b> of pERK in nuclei (marked by DAPI) of excitatory neurons revealed elevated basal nuclear pERK levels in Ptpn11^D61Y neurons. The basal levels of nuclear pERK significantly differ between the genotypes (unpaired t-test, ###p≤0.001). The stimulation of neuronal activity induces a rapid increase in the nuclear pERK level (in relation to the basal levels) in control neurons but not in those from Ptpn11^D61Y mice. The inhibition of MEK1 using SL327 for 24 h prior to the stimulation normalized the elevated basal pERK in nuclei of Ptpn11^D61Y neurons and fully restored the activity-induced increase of nuclear pERK. The identical treatment affected neither the basal nuclear pERK levels nor their stimulation-induced increase in control neurons. Data are presented as mean ± SEM; numbers in columns indicate the numbers of analyzed cells. Significance is assessed using unpaired t-test and one-way ANOVA followed by Bonferroni´s multiple comparison test (**p≤0.01, ***p≤0.001).</p

Analysis of DEGs in hippocampi of control and Ptpn11^D61Y mice in the basal state and after the stimulation of neuronal activity.

<p><b>(A)</b> The heat maps show the expression levels of DEG transcripts for each analyzed dataset. Three biological replicates (columns) per condition are shown for all DEGs (rows). The color code in a logarithmic scale is given for the signal intensities of the DEGs and indicates a low (in blue) or high (red) expression. <b>(B)</b> The Venn diagram shows the number of DEGs in each dataset. The intersections indicate the number of genes regulated in two or more datasets. <b>(C)</b> The table shows the number of differentially expressed mRNAs and miRNAs as well as the direction of their regulation in each dataset. <b>(D-F)</b> Plots show the inter-dependency of the expressional regulations of the DEGs that are commonly regulated between the datasets. Each data point represents one DEG; the x- and y-axis indicate the level of regulation as fold change in a logarithmic scale in the dataset. The correlation coefficient (r) and p-value are indicated in the graphs. The black lines show the best fits; the dashed lines indicate the 95% confidence intervals.</p

Neuronal activity-induced phosphorylation of ERK is disturbed in Ptpn11^D61Y.

<p><b>(A)</b> Exemplary photograph of an acute slice used for experiments. <b>(B)</b> Western blot of lysates from control and Ptpn11^D61Y acute slices treated with 4AP/Bic (<b>+</b>) or vehicle (<b>-</b>) for 10 minutes and probed with antibodies against pERK, ERK and β-III-tubulin. The latter was used as a loading control. <b>(C-E)</b> Quantification of the Western blot experiment as exemplified in B is shown. The stimulation of control slices led to a significant increase of the pERK level <b>(C, D)</b>. The basal pERK level was increased in Ptpn11^D61Y slices compared to controls. No further increase of pERK immunoreactivity could be detected upon stimulation of neuronal activity <b>(C, D)</b>. Note the increased total expression of ERK in the slices from Ptpn11^D61Y in the basal state and upon stimulation <b>(E)</b>. <b>(F-H)</b> The quantification of ERK phosphorylation in the nuclear fraction prepared from forebrains indicates an increase in the pERK level in the samples from Ptpn11^D61Y animals as compared to controls. Data are shown as mean ± SEM and analyzed using either one-way ANOVA followed by Bonferroni´s multiple comparison test or unpaired t-test (*p≤0.05, ***p≤0.001). The number of replicates from a total of three independent experiments is indicated in the columns of the graph.</p

The PI3K-AKT signaling is altered in Ptpn11^D61Y brains.

<p>(A) Representative Western blots of forebrain homogenate from control and Ptpn11^D61Y mice. The phosphorylation level of AKT at the residues Thr308 and Ser473 are reduced, while the phosphorylation of S6K is increased. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006684#pgen.1006684.s008" target="_blank">S8 Fig</a> for representative images of all proteins analyzed. <b>(B)</b> Quantification of the Western blots exemplified in <b>A</b>. Data is shown as mean ± SEM and normalized to the expression in controls (n = 3 animals per genotype, unpaired t-test; *p<0.05; **, p<0.01).</p