Free non-visual arrestins enhance ERK2 phosphorylation by MEK1.

<p><b>A, B.</b> ERK2 (12 pmol) was incubated with MEK1 (2 pmol) in 0.1 ml of 50 mM Hepes-Na, pH 7.2, 100 mM NaCl, and 0.1 mM [γ-³²P]ATP in the absence (control) or presence of 4.4 pmol of arrestin-2 (Arr2), arrestin-3 (Arr3), or arrestin-3-(1–393) (Arr3-(1–393)) for 30 min at 30°C. The reaction was stopped by MeOH-precipitation of the proteins. The pellet was dissolved in SDS sample buffer and subjected to SDS-PAGE. The gels were stained, dried, and exposed to X-ray film to visualize radiolabeled bands (panel <b>A</b>). ERK2 bands were cut out and ³²P incorporation was quantified by scintillation counting (panel <b>B</b>). Means ± SD of four independent experiments are shown. (**) p<0.01, as compared to control.</p

ERK2 binding to arrestin-1 and both non-visual arrestins is direct.

<p><b>A</b>. Active (phosphorylated at T183 and Y185 by MEK1) or inactive ERK2 (30 pmol) was pre-incubated with or without 30 pmol of indicated arrestin for 20 min at 30°C, then phosphorylated rhodopsin (50 pmol) was added and incubated in the light (to produce P-Rh*) in 0.1 ml for 5 min. Rhodopsin-containing membranes were pelleted through 0.2 M sucrose cushion and dissolved in SDS sample buffer. ERK2 in the pellet (1/300 of each sample) was quantified by Western blot using anti-ERK antibodies (Cell Signaling) and known amounts of purified ERK2 to generate calibration curve. Abbreviations: Arr1, visual arrestin-1, Arr2, arrestin-2, Arr3, arrestin-3. Representative blot is shown. <b>B</b>. Quantification of ERK2 binding to P-Rh*-associated arrestins. <b>C</b>. CNBr-activated Sepharose (30 µl) containing 9 µg of covalently attached active phosphorylated (without or with 1 mM ATP) or inactive ERK2 was incubated with 3 µg of indicated purified arrestin in 60 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EGTA, 1 mM DTT) for 20 min at 30°C. The beads were washed twice with 1 ml of ice-cold binding buffer supplemented with 0.01 mg/ml BSA. Bound arrestins were eluted with SDS sample buffer and quantified by Western blot, where known amounts of respective arrestins were run alongside samples to generate calibration curves. Means ± SD of three independent experiments are shown in panels <b>B</b> and <b>C</b>.</p

The effect of different β2AR ligands on ERK2 binding to arrestins and ERK2 activation.

<p>HA-tagged ERK2 was co-expressed with Flag-tagged WT arrestin-2 (<b>A,B,C</b>), or arrestin-3 (<b>D,E,F</b>) in COS-7 cells. Cells were serum starved 24 hours after transfection and stimulated for 10 min at 37°C with 10 µM of indicated β2AR ligands. Arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated ERK2 was visualized with anti-HA antibody. The binding of ERK2 to arrestin-2 (<b>B</b>) or arrestin-3 (<b>E</b>) was significantly increased by treatment with ligands. <b>C,D.</b> ERK1/2 activation in cell lysates was determined by Western blot with anti phospho-ERK1/2 antibody. Means ± SD of 3–4 independent experiments are shown in bar graphs; representative blots are shown in panels <b>A</b> and <b>D</b>. ANOVA with Bonferroni post-hoc test revealed the following differences: *, p<0.05; **, p<0.01; ***, p<0.001, as compared to untreated cells.</p

WT and Δ7 mutant of arrestin-2 rescue β2AR-mediated ERK activation in response to ICI118551 in DKO MEFs.

<p>DKO MEFs were infected with retrovirus encoding GFP (control, -), or untagged WT arrestin-2 (A2-WT), arrestin-2-3A (A2-3A), or arrestin-2-Δ7 (A2-Δ7). The cells were serum-starved 48 hours post-infection for 2 hours, stimulated with 1 µM ICI118551 for 10 min at 37°C, lysed, and analyzed by Western blot. Means ± SD of 3–4 independent experiments are shown in bar graphs; representative blots are shown below. *, p<0.05; **, p<0.01.</p

Conformational dependence of the interaction of non-visual arrestins with ERK2.

<p>COS-7 cells were transfected with WT, 3A, or Δ7 mutant forms of Flag-tagged arrestin-2 (<b>A</b>) or arestin-3 (<b>B</b>), along with ERK2-HA, with or without HA-β2AR. Cells were serum starved overnight 24 hours post-transfection and treated for 10 min at 37°C with or without 10 µM β2AR agonist isoproterenol. Cells were lysed, and arrestins were immunoprecipitated with anti-Flag antibody, and co-immunoprecipitated ERK2 and β2AR were detected with anti-HA antibody. Bar graphs show the ratio of co-immunoprecipitated ERK2 to immunoprecipitated arrestin. The data from three independent experiments were statistically analyzed by ANOVA. The significance of the differences is indicated, as follows: * or <b>^&</b>, p<0.05; ** or <b>^&&</b>, p<0.01, as compared to corresponding within group basal level of ERK2 co-immunoprecipitation (black bars); <b>^a</b> or <b>^$</b> or <b>^#</b>, p<0.05 compared to WT control (black bar in WT group).</p

Label Free Fragment Screening Using Surface Plasmon Resonance as a Tool for Fragment Finding – Analyzing Parkin, a Difficult CNS Target

<div><p>Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.</p></div

Functionally active FL-Parkin binds three different protein ligands.

<p>FL-FLAG Parkin was captured on a CM5 sensor chip with immobilized anti-FLAG antibody at a stoichiometry of (3∶1) (Parkin:Ab). Each of the protein ligands was injected at concentrations above 10-fold K_D if possible: (a) Ubiquitin at 500, 250, 125, 62.5, 31.25 and 15.62 μM (b) His-UblD and (c) UbcH7 were injected at 140, 46.7, 15.6, 5.2, 1.7 and 0.6 μM over FL-FLAG Parkin. All data was fitted to 1∶1 binding model. Each binding test was repeated at different test occasions (n≥3).1∶1 binding isotherms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066879#pone.0066879.s004" target="_blank">Figure S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066879#pone.0066879.s006" target="_blank">6</a>.</p

Thermal treated FL-FLAG-Parkin is more active than non-thermal treated FL-FLAG-Parkin independent of reducing agent.

<p>TR-FRET S5a assay using 150 nM FL-FLAG Parkin and 200nM biotinylated S5a substrate in the presence or absence of 5 mM reducing agent. FL-FLAG Parkin was incubated at 56°C for 30 min and then cooled to RT (thermal treated). Thermal treated FL-FLAG Parkin exhibit significantly different levels of Parkin activity in presence of each of the three reducing agents p<0.0001 (hatched bar) (n = 4). Non thermal treated FL-FLAG Parkin showed similar levels of activity in presence of either DTT or TCEP with p = 0.5368 and both activities are significantly higher than in presence of BME with p<0.0001 (white bar) (n = 4).</p

Parkin fragment screen data.

<p>(A) Graphical representation of a typical run of the Parkin fragment screen representing 10% of the total number of fragments screened (560 fragments). The binding level of each fragment of the run is shown in Response Units (RU) on the x-axis and the number of fragments on the y-axis. Experiments were performed on a Biacore 4000 instrument w buffer containing 2% DMSO. FL-FLAG Parkin was captured on a CM5 sensor chip with immobilized anti-FLAG antibody at a stoichiometry of (3∶1) (Parkin:Ab). Fragments were injected at 25 μM in buffer containing 2% DMSO All data were reference subtracted, solvent corrected and adjusted for changes in surface activity during a run.(B) Fragment binding levels as %Rmax of single concentration SPR hits at 25 uM of a screen of 5260 fragments. Only hits with a binding level greater than 3-fold standard deviation (SD) and acceptable sensorgrams are shown.</p

Physico-chemical properties of SPR hits vs. fragment library.

<p>The distribution of compounds in the fragment library is shown as circles; the SPR hits as triangles. The distribution of SPR hits is consistent with the fragment library (though noisier because of the small number of compounds), with the exception of hydrogen bond donors, which are over-represented in the SPR hits compared to fragment library.</p