Dynamic rearrangement of cell states detected by systematic screening of sequential anticancer treatments

Modeled data generated by a Bayesian dose-response framework predicting sequentially effective drug combinations from an oncological drug matrix screen, comprising 10,000 drug combinations in melanoma and pancreatic cancer cell lines

Schematic of the Linear Motif Discovery Strategy

<p>Interaction maps are probed for interaction sets (A): Partners of proteins with multiple interactions are clustered together when there are no known sequence features present (B). Domains and homologous regions are then identified (B) and removed prior to running exhaustive pattern discovery (C) to produce a list of motifs ranked by their probabilities <i>P</i> (D). Hypothetical motifs are shown as coloured squares in (C) and (D). “Proteins” in (D) gives the set of proteins containing at least one copy of the motif.</p

A Lit-1 MAP Kinase SxPxxxS Motif

<p>The MAP kinase lit-1 surrounded by its interaction partners containing the SxPxxxS motif. Details are as for <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-g003" target="_blank">Figure 3</a>. Yellow boxes show the location of deletion mutants known to affect the interaction. Cbr, <i>C. briggsae;</i> Cel, <i>C. elegans</i>.</p

An Acidic Yeast PP1 Binding Motif

<div><p>(A) PP1 (Glc7) with the set of interaction partners containing the DxxDxxxD motifs. Details are as for <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-g003" target="_blank">Figure 3</a>A. Here the location of RVxF motifs (defined as matches to (RK)x_0–1(VI)x(FW)) are shown as yellow bars, and low-complexity regions are magenta. The figure also shows the structure of PP1 bound to RVxF (red spheres) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b54" target="_blank">54</a>] with a hypothetical helix containing the motif. Blue spheres show the location of Arg or Lys residues, and the active site is circled with critical Arginines shown in ball-and-stick. Red arrows show hypothetical interactions of the motif either with sites on PP1 or elsewhere. Ani, Aspergillus nidulans; Cal, Candida albicans; Ego, Eremothecium gossypii; Gze, Gibberella zeae; Mgr, Magnaporthe grisea; Sce, S. cerevisiae; Spo, Schizosaccharomyces pombe; Str, Salinospora tropicalis; Uma, Ustilago maydis; Xla, Xenopus laevis.</p> <p>(B) Saturation curves, showing bound fraction as a function of PP1 concentration. The polarization values (mP) were normalized to an extrapolated B_max because B_max could not be reached experimentally. Other details are as given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-g003" target="_blank">Figure 3</a>B. Red triangles in the lower panel show the location of the near match to the motif in the mutated sequence.</p></div

A Novel Fly VxxxRxYS Motif That Binds Translin

<div><p>(A) Translin (left) shown surrounded by interaction partners containing the predicted motif VxxxRxYS. Proteins are shown as lines with domains (labelled shapes), predicted coiled coils (light blue/green segments), and the location of motifs (blue vertical bars). Sequences for the motif-containing region are shown aligned to the best homologues in closely related species. Amino acids are coloured according to residue type: blue, positive; red, negative; light blue, small; yellow, hydrophobic; green, aromatic; magenta, polar; and orange, proline. Those constituting the predicted motif are denoted by circles. Aga, <i>Anopheles gambiae;</i> Dme, <i>D. melanogaster;</i> Dps, <i>D. pseudoobscura</i>.</p> <p>(B) Saturation curves, showing bound fraction (fluorescently labelled peptides at saturation) as a function of Translin concentration. Polarization values (mP) at zero concentration and B_max were normalised to give the bound fraction. <i>K</i>_D was computed by non-linear regression on values from three independent experiments. The lower panel shows the alignment of the native and mutated peptides together with the arbitrary peptide (selected randomly). Black triangles show positions specifying the motif (VxxxRxYS). The alignment is coloured as described in (A).</p></div

Overview of Motifs Found in the Fly

<p>Significant predictions from the yeast two-hybrid set for the fly. Blue dots in the center of each cluster represent proteins with four or more interaction partners (red and white dots) containing at least one confidently predicted motif (<i>p-</i>value < 0.001; <i>S_cons</i> ≤ 8 × 10⁻¹⁵). Partner proteins containing the motif are represented by red dots, whereas proteins lacking the motif are indicated by white dots. Clusters are labelled as gene name→detected motif. Yellow circles enclose known motifs: SH3→PxxP [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b38" target="_blank">38</a>], PP1→RVxF [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b22" target="_blank">22</a>], C-terminal binding protein (CtBP)→PxDLS [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b52" target="_blank">52</a>], SR splicing factors RS-rich segments [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b53" target="_blank">53</a>], and CG6843→SxKSKxxK, a likely nuclear localization signal. The Translin→VxxxRxYS motif was experimentally tested (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-g003" target="_blank">Figure 3</a>). The grey circles enclose clusters with low-complexity patterns. Two additional known motifs were also found in the fly using more relaxed criteria than those used for the other motifs in the figure: Groucho→WRPW [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b07" target="_blank">7</a>] and Dynein light chain→TQT [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030405#pbio-0030405-b26" target="_blank">26</a>] as the variant A(TI)QT(DE). The latter was also identified as significant in the domain sets. Proteins are denoted either by their FlyBase accession codes or protein names when available.</p

Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed

Signaling through the Hippo pathway by re-engineered PAK4.

(A) Phosphorylation of LATS and YAP in parental HEK293A (WT) or a derivative lacking eight STE20 family kinases (MM8-KO) were analyzed by immunoblotting following transfection with plasmids expressing the indicated kinases. (B) Quantified Rel. PIs from immunoblots were normalized to the signal from WT cells (n ≥ 7, error bars indicate SD). Student t tests were used to determine whether the indicated pairs were significantly different from each other (*p p F = 34.06, p p p = 0.05). Error bars represent 95% CI. Numerical data for (B) and (D) are provided in S2 Data. DAPI, 4′,6-diamidino-2-phenylindole; EV, empty vector; GEF-H1, Rho guanine nucleotide exchange factor H1; HEK, human embryonic kidney; LATS, large tumor suppressor homolog; MST, Mammalian sterile 20 kinase; PAK, p21-activated kinase; P-, phospho-; Rel. PI, relative phosphorylation index; SE, standard error; WT, wild type; YAP, Yes-associated protein.</p

Phosphorylation kinetics of peptide substrates by STE20 kinases.

(A) Michaelis–Menten curve for MST4 phosphorylation of MSTtide. Individual data points from three separate experiments are shown. (B) Initial rates of phosphorylation by MST4 of a series of peptides with the indicated sequences (n ≥ 3, bars show mean ± SD) shown relative to MSTtide (top) phosphorylation. Data for PAKtide are at bottom. (C) Relative initial rates of phosphorylation of MSTtide and PAKtide by a series of STE20 kinases (n = 3, error bars are 95% CI). Kinases with bars that do not cross the y-axis have a statistically significant preference for one consensus peptide over the other (p = 0.05). Source data for all panels are provided in S2 Data. GCK, germinal center kinase; HGK, HPK/GCK-like kinase; HPK1, Hematopoietic progenitor kinase 1; KHS, Kinase homologous to SPS1/STE20; LOK, Lymphocyte-oriented kinase; MINK, Misshapen-like kinase 1; MST, Mammalian sterile 20 kinase; MYO, myosin; OSR1, Oxidative stress-responsive 1; PAK, p21-activated kinase; SLK, STE20-like kinase; TAO, thousand and one amino acid kinase; YSK1, Yeast Sps1/Ste20-related Kinase 1</p

Acidic residues in the β3–αC loop region promote selection of basic residues by PKCβ.

PSPA analysis shown as heat maps and sequence logos (prepared as in Fig 1) for WT (A) and the β3–αC loop mutant (B) of PKCβ. Numerical data are provided in S1 Data. PKC, protein kinase C; PSPA, positional scanning peptide array; WT, wild type.</p