Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: Human A431 cells were stimulated with EGF and dynamic responses analyzed using three complementary methods. Samples snap-frozen at different time-points after EGF treatment were analyzed by mass spectrometry (MS) to detect relative changes over time in abundance of specific tyrosine-phosphorylated peptides. Parallel samples were analyzed by far-western blotting with a panel of SH2 domain probes to visualize changes over time in binding sites for each SH2 domain. Finally, living cells expressing fluorescent SH2 domains were imaged to quantify membrane binding dynamics for each SH2 domain. DOI: http://dx.doi.org/10.7554/eLife.11835.003

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Mass Spectrometry, Far Western Blot, Binding Assay, Expressing, Membrane

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative anti-pY Western (upper panel) and far-Western blots (next four panels) of 60-min EGF stimulation time-course. Far-Westerns using GRB2, SHP2-N, NCK1 and RASGAP-N are shown to illustrate major binding patterns identified (see B ). Additional SH2 blot data are provided in . Immunoblotting with antibodies to EGFR and tubulin was used to confirm equal loading. ( B ) Hierarchical clustering of SH2 domains on the basis of binding to four major phosphoproteins (EGFR, GAB1, p130CAS, and SHCA). Signal was normalized to maximum band intensity across all time points and all bands for each probe replicate. Then data for each phosphoprotein was averaged in a probe specific manner (red represents greater percentage of total signal, max = 1, min = 0). Names of SH2/PTB domain probes are indicated on the right. Colored boxes represent SH2 clusters defined by un-centered correlation coefficient >0.85. DOI: http://dx.doi.org/10.7554/eLife.11835.004

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Western Blot, Binding Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: Protein names are labeled on the left and stimulation times are on top. For probe names, '-N, -C, or -NC' indicates the use of N-terminal, C-terminal, or natural tandem SH2 domain probes. See for construct information. DOI: http://dx.doi.org/10.7554/eLife.11835.005

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Labeling, Construct

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Hierarchical clustering of relative SH2 specificity. Values were obtained by subtracting percent bound by pY from percent bound by each SH2 for each time point. Red represents binding greater than pY and green represents binding less than pY. Relative SH2 specificity value = %Band intensity SH2 at time x - %Band intensity anti-pY at time x. ( B ) Hierarchical clustering of SH2 binding to EGFR band (normalized to maximum, max=1, min=0) for 60-min time-course. ( C ) Hierarchical clustering of EGFR phosphosite-specific Western blotting quantifications (normalized to maximum, max=1, min=0) for 60-min time-course. ( D ) and ( E ) Plots comparing the EGFR phosphosite kinetics and SH2 binding kinetics for SH2 domains and their canonical binding motifs ( D , CRKL and pY992, R 2 =0.81; E, GRB2 and pY1168, R 2 =0.91). ( F ) Plot comparing total EGFR phosphorylation (pY EGFR band) with SHCA PTB binding (R 2 =0.89). R-squared values were calculated by plotting normalized pY Western intensity against normalized SH2 binding FW intensity. pY EGFR quantifications consist of data from a single experiment.( G ) pERK1 and pERK2 pY/pT activation site phosphorylation kinetics following stimulation with EGF. Lines represent average of two technical replicates. DOI: http://dx.doi.org/10.7554/eLife.11835.007

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Binding Assay, Western Blot, Activation Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative total internal reflection fluorescence (TIRF) microscopy images of fluorescently tagged SH2 domains before and 40 min after EGF stimulation, for SHP2-N, GRB2, NCK1 and RASGAP-N SH2 domains. Scale bars = 10 μm. ( B ) Comparison of change in total membrane SH2 fluorescence from imaging live cells ( red ) and change in FW-based SH2 binding ( black ) following EGF stimulation. Gray lines indicate TIRF background signal. Data is normalized to maximum. See for complete dataset. FW data represent average of multiple technical replicates; in vivo data are from single representative experiments. DOI: http://dx.doi.org/10.7554/eLife.11835.010

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Fluorescence, Microscopy, Comparison, Membrane, Imaging, Binding Assay, In Vivo

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: Quantification of in vivo SH2 binding dynamics and binding site kinetics. To compare in vivo imaging and FW data directly, for each SH2 domain in vivo membrane recruitment kinetics and timecourse of total binding (to all bands) by FW were fit to the first order exponential recovery function (1-e -t/τ ), where τ is the time constant and t is time after EGF. *denotes data that fit poorly to the recovery function (R-square <0.5). n = number of biological replicates used for calculation of τ from in vivo imaging. SEM of τ values from multiple replicates shown in parentheses. D is the diffusion constant of SH2 molecules on the membrane in cells stimulated by EGF, measured by single-molecule tracking technique 40 min post-EGF (see Materials and methods). For each SH2, data from >3000 trajectories in a single cell were used to calculate D. ND = not determined. DOI: http://dx.doi.org/10.7554/eLife.11835.019

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: In Vivo, Binding Assay, In Vivo Imaging, Membrane, Diffusion-based Assay, Imaging, In Vitro

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A-C ) TIRF images of additional fluorescently tagged SH2 domains before and after EGF stimulation. A) GAB1 binding domains (SHP2-NC) ( B ) EGFR binding domains (GRB7) and ( C ) p130CAS binding domains (CRK, RASGAP-NC). Domains are labeled according to clustering results from . Post-EGF images were taken ~40min after stimulation. Scale bars = 10 μm ( D ) Correlation plot of SH2 domain probe diffusion rate ( D , y-axis) and recruitment time constant (τ, x-axis). Data for SHP2-C was an outlier and was removed from the plot for clarity. Error bars for τ values represent SEM (see ). DOI: http://dx.doi.org/10.7554/eLife.11835.011

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Binding Assay, Labeling, Diffusion-based Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative white light ( top panel ) and epifluorescence images ( lower panel ) of GRB2 SH2-tdEOS transfected A431 cells that were used to determine total cell number, transfection efficiency and relative expression level. Insert ( upper panel) shows representative DIC image of nonadherent cells used to determine cell volume. ( B ) Histogram of individual cell GRB2 SH2-tdEOS expression levels. Left skew in expression was compensated for in the final calculation. ( C ) Anti-GRB2 SH2 blot used to calculate the average concentration of GRB2 SH2-tdEOS (6.5 μM) and endogenous GRB2 (1.5 μM). Concentrations were determined by using bacterially produced GST-GRB2 SH2 fusion as standard (right side of the blot). ( D ) Anti-pY blot showing EGF-induced EGFR phosphorylation and phosphorylation standard titration used to calculate the cellular concentration of phosphorylated EGFR sites. Concentrations were determined using a highly phosphorylated recombinant ABL standard with a known pY concentration (right side of the blot). ( E ) Representative z-axis cross-sections of fixed A431 cells immunostained with anti-pY. The images and traces were obtained from the same cell along the x- and y-axes. White block indicates the quantified area. Curves represent an average of multiple line scan quantifications across an individual cell membrane. ( F ) Apical and basal pY levels following EGF stimulation as measured by immunofluorescence. Intensity measurements were averaged from two independent experiments; a total of at least seven cells were quantified for each time point. Error is SEM for all data points. ( G ) Ratio of apical to basal phosphorylation following stimulation with EGF. DOI: http://dx.doi.org/10.7554/eLife.11835.013

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Transfection, Expressing, Concentration Assay, Produced, Titration, Recombinant, Blocking Assay, Membrane, Immunofluorescence

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Anti-pY Western and GRB2 FW of serially diluted lysates from A431 cells stimulated with 25ng/mL EGF for 0, 1.5 and 10 min. Total micrograms of lysate protein run for each lane is listed above the lane. ( B ) Fold increase in quantified GRB2 FW signal for each amount of lysate (compared to signal at 1.25 μg of lysate). For reference, all pY and FW values shown in were quantified from blots run at 20 μg/lane. DOI: http://dx.doi.org/10.7554/eLife.11835.014

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Western Blot

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative anti-pY immunoblot (upper panel) and Grb2 SH2 far-western blot (lower panel) of A431 cells stimulated with 1 ng/mL EGF and flash frozen at 22 discrete time points. ( B ) Quantification of EGFR tyrosine phosphorylation kinetics (from anti-pY immunoblot) in A431 cells treated with 1 ng/mL EGF (n=2 biological replicates) and 25 ng/mL EGF (n=3 biological replicates). ( C ) Anti-pY immunoblot of A431 cells stimulated with 1 ng/mL and 25 ng/mL EGF at 0, 1.5 and 10 min. EGFR phosphorylation was 5.4 +/- 0.4 fold greater in cells stimulated with 25 ng/mL (normalized for EGFR expression, error = SEM). No difference was observed in prestimulation EGFR phosphorylation after normalization. ( D ) Comparison of Grb2 SH2 binding site phosphorylation kinetics (GST-Grb2 SH2 FW, black; time constant τ = 54.6 +/- 1.4 s, n=2 biological replicates) and Grb2 SH2 in vivo membrane recruitment kinetics (tdEOS-GRB2 SH2 TIRF, red; time constant τ = 116.7 +/- 2.3 s, n=2 biological replicates). DOI: http://dx.doi.org/10.7554/eLife.11835.016

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Western Blot, Far Western Blot, Expressing, Comparison, Binding Assay, In Vivo, Membrane

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: sptPALM images of tdEOS-labeled p130CAS and NCK1 SH2-tdEOS in A431 cells before EGF stimulation. Scale bars = 10 μm DOI: http://dx.doi.org/10.7554/eLife.11835.017

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Labeling

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) SptPALM measurement of apparent membrane recruitment rate (γ on , black line ) and apparent membrane dissociation rate (λ off , red line ) following stimulation with EGF. γ on is computed as the total number of observed recruitment events divided by the timespan (0.8 min). λ off is computed based on the membrane dwell-times of the observed molecules as previously described . Errorbars on λ off represent statistical sampling errors due to the finite numbers of single-molecule trajectories used for this calculation. ( B ) SH2 membrane binding curve calculated using experimentally determined γ on and λ off values. ( C ) Kinetics of GRB2 SH2 binding site clustering (cluster size x cluster number) after EGF treatment. ( D ) Number of newly recruited GRB2 SH2 molecules ( black, whole ), and those within clusters ( red, cluster ) and outside of clusters ( blue, non-cluster ) after EGF stimulation. Dotted lines show fit with exponential recovery function. DOI: http://dx.doi.org/10.7554/eLife.11835.021

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Membrane, Sampling, Binding Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: pervanadate (PV) treated cells. Representative GRB2 SH2 far-Western and anti-pY (PY100) immunoblots for EGF and PV treated cells. Immunoblotting with antibodies to EGFR and actin was used to confirm equal loading. DOI: http://dx.doi.org/10.7554/eLife.11835.022

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Western Blot

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) TIRF microscopy images of fluorescently tagged GRB2 SH2 in pervanadate-treated (PV)- and EGF-stimulated cells (40 min post-stimulation). Scale bars = 10 μm. ( B ) Mean square displacement (MSD) of GRB2 SH2 in EGF- ( black ) and PV-treated cells ( red ). Results based on ~8000 (PV) and ~10,000 (EGF) pooled single-molecule trajectories from two cells. Error bars represent SEM. ( C ) Temporal progression of Grb2 SH2 recruitment rates (apparent on-rate) for two PV-treated cells. ( D ) Comparison of GRB2 SH2 recruitment kinetics (TIRF fluorescence imaging, n=2, and GRB2 FW (n=2) total binding, in either EGF-treated (in vivo) or PV-treated cells (in vivo). FW data is normalized so that the maximum signal in EGF treated cells equals 1. DOI: http://dx.doi.org/10.7554/eLife.11835.023

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Microscopy, Comparison, Fluorescence, Imaging, Binding Assay, In Vivo

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: CRK SH2 shows little or no binding to the EGFR band in COS1 cells. DOI: http://dx.doi.org/10.7554/eLife.11835.018

Article Snippet: GRB2 SH2-tdEOS and GST-GRB2-SH2 were detecting using mouse anti-GRB2 SH2 (R&D Systems, #669604). pERK1 and pERK2 were detected using rabbit anti p44/42 pT202/pY204 (CST, #9101S).

Techniques: Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: Nilotinib causes partial dissolution of BCR–ABL signaling complex. a 293T cells were transfected with p190 BCR–ABL, native cell lysates were subjected to ultracentrifugation in the 15–40% sucrose gradient, and collected fractions were analyzed by western blot. The presence of BCR–ABL signal in more than one fraction suggests the existence of complexes of different compositions. Note the various degrees of co-sedimentation of BCR–ABL with p85a-PI3K, GRB2, SHIP2, SHC1, SOS1, SHP2 and cCBL; no co-sedimentation with CRK, CRKL or GAB2 was found. Inhibition of BCR–ABL kinase activity with 100 nM nilotinib resulted in a shift of a fraction of the BCR–ABL complexes towards lighter fractions, suggesting partial dissolution of the BCR–ABL signaling complex. b The western blot analysis of proteins co-sedimenting with BCR–ABL (p85a-PI3K, GRB2 and SHIP2) was quantified as described in “Materials and methods”. Note that portion of GRB2, but not SHIP2 or p85a-PI3K dissociated from the BCR–ABL complex after nilotinib treatment. Data represent a single experiment out of three independent experiments carried out. The fractions containing most of the p190 BCR–ABL are highlighted in red. Phosphorylation (p) at ABL Y412 was used to determine the degree of BCR–ABL inhibition using nilotinib; actin serves as a loading control in total cell lysates used for ultracentrifugation. c Cells were transfected with FLAG-tagged p190 BCR–ABL, V5-tagged GRB2 or SHIP2, treated with nilotinib, and subjected to PLA. The antibodies against protein tags were used in PLA (red); cABL antibody was used to counterstain the transfected cells (green). Cells transfected with BCR–ABL and an empty vector serve as the negative control. Number of PLA dots per cell was calculated and graphed (10–90 percentile). Statistically significant differences were highlighted (Student’s t test with Welch’s correction for unequal variances; *p < 0.05, **p < 0.01). Scale bars, 10 µm

Article Snippet: GRB2 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Dissolution, Transfection, Western Blot, Sedimentation, Inhibition, Activity Assay, Plasmid Preparation, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: SHIP2 and GRB2 associate with kinase-inactive BCR–ABL. a Scheme of used experimental procedure comprising native lysis, blue-native (BN)-PAGE, SDS-PAGE and western blot. The three members of protein complex are highlighted in color. b Cell lysates of 293T cells transfected with p210 BCR–ABL. KD, kinase-dead BCR–ABL; Y177, BCR–ABL Y177F mutant. The inhibition of BCR–ABL kinase activity by nilotinib is demonstrated by the lack of autophosphorylation (p) at Y412. Actin serves as loading control. No trans, non-transfected cells. c Merged second dimension BN-PAGE blots of cells transfected with BCR–ABL variants. The membranes have been probed sequentially for BCR–ABL, SHIP2 and GRB2, the BCR–ABL/SHIP2/GRB2 complexes are highlighted by yellow box. (D) Quantification of the percentage of bound GRB2 and SHIP2 to the BCR–ABL. Statistically significant differences are highlighted (Student’s t test, **p < 0.01; n.s., not significant). Data are representative of three independent experiments

Article Snippet: GRB2 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Lysis, SDS Page, Western Blot, Transfection, Mutagenesis, Inhibition, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: The BCR–ABL signaling complex is preserved after nilotinib treatment. 293T cells were transfected with p190 and p210 BCR–ABL alone (a) or together with STS1 (b), CRKL (c) and GRB2 (d). BCR–ABL was immunoprecipitated (IP) and binding of interaction partners was analyzed by western blot. The SHC1 isoforms are indicated (p46, p52, p66). Empty, transfection with empty plasmid. BCR–ABL kinase activity was determined by detecting autophosphorylation (p) at Y412. Note the co-immunoprecipitation of SOS1, SHIP2, cCBL, SHC1, STS1, CRKL and GRB2 with BCR–ABL in cells treated with nilotinib (green arrows). Also note the co-immunoprecipitation of STS1, CRKL, and GRB2 with kinase-dead (KD) BCR–ABL (blue arrows). Data are representative of three independents experiments (n = 3). Actin serves as a loading control in cell lysates used for IP

Article Snippet: GRB2 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Transfection, Immunoprecipitation, Binding Assay, Western Blot, Plasmid Preparation, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Elucidation of protein interactions necessary for the maintenance of the BCR–ABL signaling complex

doi: 10.1007/s00018-019-03397-7

Figure Lengend Snippet: Interaction of GRB2, SOS1, cCBL, and SHC1 with BCR–ABL. a Secondary structure prediction of p210 BCR–ABL by IUPRED. Values above 0.5 indicate disordered regions IDR1 and IDR3 on the BCR–ABL N- and C-termini, involving Y177 and three NLS, respectively. Smaller disordered region IDR2 is located between domains PH and SH3. b Scheme of the microarray analysis. Thirteen amino acid long peptides corresponding to the primary sequence of p210 BCR–ABL were spotted on microarrays, incubated with protein of interest, primary and fluorescently labeled secondary antibodies, and scanned. Fluorescence intensity values for each spot were used to indicate the binding of protein to BCR–ABL peptides. c Microarrays indicate direct binding of GRB2 to phosphorylated Y177. Red lines on BCR–ABL scheme indicate potential binding sites. Graph shows averaged relative intensities for phosphorylated (red) and non-phosphorylated peptides involving peptides with Y177. Error bars indicate SD from three technical replicates shown in Fig. S2. d Co-immunoprecipitation (Co-IP) of BCR–ABL with GRB2 after expression in 293T cells; Y177F substitution abrogates GRB2 association with BCR–ABL as well as deleting the region (construct BT, bottom arrows). Side arrows indicate electrophoretic mobility shift GRB2 phosphorylated by BCR–ABL. e, f Co-immunoprecipitation of endogenous cCBL, SHC1 and SOS1 with transfected BCR–ABL in 293T cells. Please note the compromised SHC1 binding on BCR–ABL–BT, -ΔST, -ΔTK and KD variants (blue arrows). Y177F abrogates binding of SOS1 and largely limits the binding of cCBL (green arrows). Data are representative of three independent experiments (n = 3). g Quantification of SHC1 co-IP with BCR–ABL constructs from (f). SHC1 was normalized to BCR–ABL levels, error bars indicate SD from four independent experiments. Statistically significant differences are indicated (Student’s t test, *p < 0.05, ***p < 0.001; ns non-significant). h Scheme of the proposed interaction. GRB2 binds directly to phosphorylated Y177 and recruits SOS1. cCBL also requires GRB2 for recruitment. SHC1 requires TK domain and pleckstrin homology (PH) domain for binding

Article Snippet: GRB2 (RC200469), p85a-PI3K (RG210544) and SHIP2 (RC214716) vectors were obtained from Origene, CRKL (HG11261-CH) and STS1 (HG13868-NF) vectors were obtained from Sino Biological.

Techniques: Microarray, Sequencing, Incubation, Labeling, Fluorescence, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Construct, Electrophoretic Mobility Shift Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking *

doi: 10.1074/jbc.M116.762377

Figure Lengend Snippet: Acute removal of plasma membrane cholesterol induces recruitment of endophilin-A2, endophilin-B1, and SphK1 to tubular invaginations. A, live-cell confocal images of H460 cells expressing endophilin-A2-GFP, SphK1-GFP, or endophilin-B1-GFP, as indicated, before and 320 s after 10 mm MβCD treatment at 37 °C. A total of four independent experiments with a minimum of 5 cells/experiment were performed, and representative images are shown. B and C, confocal images of H460 cells expressing endophilin-A2-GFP and V5-tagged SphK1 (B) or endophilin-B1-GFP and V5-tagged SphK1 (C) before and 360 s after 10 mm MβCD treatment at 37 °C and immunostained with anti-V5 (purple). Arrowheads point to foci formed after MβCD treatment where endophilins and V5-tagged SphK1 co-localize. Scale bars = 10 μm.

Article Snippet: Plasmids, siRNA, Transfection, and Overexpression The endophilin-A2-GFP (catalog no. RG201552) and endophilin-B1-GFP (catalog no. RG200106) plasmids were from Origene, and control scrambled siRNA and siRNA targeting caveolin-1 (catalog no. L-003467) were from Thermo Fisher Scientific.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking *

doi: 10.1074/jbc.M116.762377

Figure Lengend Snippet: SK1-I induces recruitment of SphK1, endophilin-A2, and endophilin-B1 to vesicles and dynamic filamentous structures. A–E, live-cell confocal images of H460 cells expressing SphK1-GFP (A and E), endophilin-A2-GFP (B), or endophilin-B1-GFP (C and D) treated with 10 μm SK1-I for 45 min. D, bottom panels, time-lapse (30-s intervals) images of the region outlined in red. Zoom, magnification of SphK1-GFP-positive (A) or endophilin-A2-GFP-positive (B) vesicles and endophilin-B1-GFP positive filaments (C). Arrowheads in C point to filaments that are contiguous with vesicles. Arrowheads in E point to SphK1-positive filaments. Scale bars = 10 μm.

Article Snippet: Plasmids, siRNA, Transfection, and Overexpression The endophilin-A2-GFP (catalog no. RG201552) and endophilin-B1-GFP (catalog no. RG200106) plasmids were from Origene, and control scrambled siRNA and siRNA targeting caveolin-1 (catalog no. L-003467) were from Thermo Fisher Scientific.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking *

doi: 10.1074/jbc.M116.762377

Figure Lengend Snippet: Relocalization of endogenous endophilin-A2 and endophilin-B1 following treatment with SK1-I and sphingosine. A, C, and D, confocal images of H460 cells treated with vehicle (A), 10 μm SK1-I for 30 min (A and D), or 1 μm PF543 for 30 min followed by 10 μm Sph (C) for 30 min. Cells were then immunostained with anti-endophilin-A2 (red) or anti-endophilin-B1 (green), and nuclei were labeled with Hoechst. Arrowheads in A point to filamentous structures. A and C, zoom, magnification of double-positive foci (A, top) and vesicles (A, bottom, and C). B, the diameters of foci and vesicles in A were determined. D, 3D reconstructions of cells treated without or with 10 μm SK1-I for 30 min. A total of 37 0.44-μm slices were collected for each channel. One slice in the right panel was used in A. Data are mean ± S.D. ***, p ≤ 0.001. Scale bars = 10 μm.

Article Snippet: Plasmids, siRNA, Transfection, and Overexpression The endophilin-A2-GFP (catalog no. RG201552) and endophilin-B1-GFP (catalog no. RG200106) plasmids were from Origene, and control scrambled siRNA and siRNA targeting caveolin-1 (catalog no. L-003467) were from Thermo Fisher Scientific.

Techniques: Labeling

Journal: PLoS ONE

Article Title: Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

doi: 10.1371/journal.pone.0102899

Figure Lengend Snippet: GST-hADAM10(697-748)-precipitated SH3 domains: Function and localization of putative interaction partners according to the UniProt Protein Knowledgebase (UniprotKB).

Article Snippet: To verify the interaction of ADAM10 with Endophilin-A2/EEN, murine ADAM10 was transfected alone or in combination with a human Endophilin-A2/EEN expression construct (SKU: SC118256) obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Binding Assay, Migration, Transmission Assay

Journal: PLoS ONE

Article Title: Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

doi: 10.1371/journal.pone.0102899

Figure Lengend Snippet: ( A ) In order to verify the potential interaction of ADAM10 with Endophilin-A2/EEN, HEK 293T cells were either left untransfected or transfected with HA-tagged murine ADAM10 alone or in combination with human Endophilin-A2/EEN. 18 h later, the cells were lysed and immunoprecipitations (IPs) were performed with monoclonal antibodies directed against the HA-tag (clone 3F10) or EEN (clone 2F5), respectively. Protein input for IPs was 1.8 or 2 mg of protein, respectively. Of note: at the employed exposure time, endogenous EEN is hardly detectable in the whole cell lysates containing a total of 10 µg of protein. ( B ) Pull down analyses were performed from PHA blasts (day 16) using a GST fusion protein containing the SH3 domain of EEN coupled to GST (EEN SH3) and GST as a control. The subsequent Western blot was probed with anti-ADAM10 (clone 11G2).

Article Snippet: To verify the interaction of ADAM10 with Endophilin-A2/EEN, murine ADAM10 was transfected alone or in combination with a human Endophilin-A2/EEN expression construct (SKU: SC118256) obtained from OriGene Technologies (Rockville, MD, USA).

Techniques: Transfection, Western Blot

Journal: Journal of the American Chemical Society

Article Title: High-Affinity Interactions of the nSH3/cSH3 Domains of Grb2 with the C-Terminal Proline-Rich Domain of SOS1

doi: 10.1021/jacs.9b10710

Figure Lengend Snippet: Grb2 and SOS1 PR domain. (A) Full-length human Grb2 (PDB: 1GRI) contains 217 residues and is composed of nSH3 (blue, residues 1–58), SH2 (white, residues 60–153), and cSH3 (green, residues 156–215) domains. The polar, nonpolar, positively charged, and negatively charged residues are colored green, black/white, blue, and red, respectively, in the amino acid sequence of Grb2 (top). Available NMR/crystal structures of nSH3/cSH3 domains with the binding partners are nSH3–VPPPVPPRRR (PDB: 1AZE) and cSH3–APPPRPPKP (PDB: 2W0Z). (B) Candidate nSH3/cSH3 binding sites on SOS1 are denoted as S1–S10 in the PR domain (residues 1014–1333) amino acid sequence, colored the same way as for Grb2.

Article Snippet: Wild-type human Grb2 nSH3/cSH3 plasmids and selected SOS1 PR peptides were commercially obtained from Biomatik Corporation.

Techniques: Sequencing, Binding Assay

Journal: Journal of the American Chemical Society

Article Title: High-Affinity Interactions of the nSH3/cSH3 Domains of Grb2 with the C-Terminal Proline-Rich Domain of SOS1

doi: 10.1021/jacs.9b10710

Figure Lengend Snippet: The four most probable structural models of the Grb2–SOS1 complex. Grb2–SOS1 interactions are suggested by the strong binding affinity of (A) nSH3–S4/cSH3–S10 (mode 1), (B) nSH3–S5/cHS3–S4 (mode 2), (C) nSH3–S5/cHS3–S10 (mode 3), and (D) nSH3–S9/cHS3–S4 (mode 4). The models of Grb2–SOS1 PR are obtained from replica exchange simulation for 100 ns. Grb2 nSH3, SH2, cSH3, and SOS1 PR domain are colored blue, white, green, and yellow, and the binding peptides (S4, S5, S9, and S10) are shown as red tubes. The heat maps of contact frequency show the interactions between nSH3/cSH3 residues (y-axis) and various SOS1 peptides residues (x-axis) during the simulation. The dark brown color indicates highly frequent contacts. The three main interaction regions of nSH3/cSH3 are around residues 10, 35, and 50 for nSH3 and 170, 195, and 205 for cSH3. These maps show correlation between nSH3–SOS1 and cSH3–SOS1 interactions. For mode 2, the unfolded S5 tends to dissociate from nSH3, resulting in unstable Grb2–SOS1 association.

Article Snippet: Wild-type human Grb2 nSH3/cSH3 plasmids and selected SOS1 PR peptides were commercially obtained from Biomatik Corporation.

Techniques: Binding Assay

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Modular organization of Grb2 adaptor and Gab1 docker. (a) Grb2 is comprised of a central SH2 (Src homology 2) domain flanked between an N-terminal SH3 (nSH3) domain and a C-terminal SH3 (cSH3) domain. The amino acid sequence of the cSH3 domain is indicated with the residues constituting the β1–β6 strands clearly demarcated. Key amino acid residues within the cSH3 domain involved in recognition of cognate ligands are colored red and labeled for clarity. (b) Gab1 is constructed on an N-terminal PH (Pleckstrin homology) domain and a C-terminal proline-rich (PR) domain separated by a long stretch of uncharacterized region. The PR domain contains four distinct RXXK motifs, here designated G1, G2, G3 and G4. The amino acid sequence of these motifs and flanking residues within Gab1 is provided. The numbering of various residues within and flanking the RXXK motifs is based on the nomenclature suggested by Feller and co-workers (Harkiolaki et al., 2009).

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Sequencing, Labeling, Construct

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Representative ITC isotherms for the binding of cSH3 domain of Grb2 to Gab1-derived peptides G1 (a), G2 (b), G3 (c) and G4 (d). The upper panels show the raw ITC data expressed as change in thermal power with respect to time over the period of titration. In the lower panels, change in molar heat is expressed as a function of molar ratio of corresponding Gab1 peptide to cSH3 domain of Grb2. The solid lines in the lower panels show the fit of data to a one-site model, as embodied in Eq [1], using the ORIGIN software. Note that all data are shown to same scale for direct comparison.

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Derivative Assay, Titration, Software, Comparison

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Thermodynamic parameters for the binding of cSH3 domain of Grb2 to wildtype and various mutant Gab1-derived peptides

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Mutagenesis, Sequencing

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Thermodynamic parameters for the binding of cSH3 domain of Grb2 to single alanine mutants of Gab1-derived G1 peptide

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Sequencing

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Thermodynamic parameters for the binding of cSH3 domain of Grb2 to single alanine mutants of Gab1-derived G2 peptide

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Sequencing

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Thermodynamic parameters for the binding of Gab1-derived peptides G1 and G2 to wildtype (WT) and various mutant cSH3 domains of Grb2

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Mutagenesis

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: Effect of salt on the energetics of binding of cSH3 domain of Grb2 to Gab1-derived peptides G1 and G2 as analyzed by ITC. (a) lnKd versus ln[NaCl] plots (upper panel), Kd versus [NaCl] plots (middle panel) and ΔGion versus [NaCl] plots (lower panel) for the binding of cSH3 domain to G1 peptide (●) and G2 peptide (▲). In the upper panel, the solid lines show linear fits to the data points, while solid lines in the middle and lower panels are merely used to connect data points for clarity. (b) Dependence of thermodynamic parameters ΔH, TΔS and ΔG on [NaCl] for the binding of cSH3 domain to G1 peptide (●) and G2 peptide (▲). The solid lines in all panels are merely used to connect data points for clarity. Each data point is the arithmetic mean of 3–4 independent experiments. All error bars are given to one standard deviation.

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Derivative Assay, Standard Deviation

Journal:

Article Title: Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

doi: 10.1002/jmr.1080

Figure Lengend Snippet: 3D structural models of the cSH3 domain of Grb2 in complex with Gab1-derived peptides G1 and G2. (a) Ribbon representation of the cSH3 domain bound to G1 peptide. The β-strands in the cSH3 domain are shown in yellow with loops depicted in gray and the sidechains of key residues involved in making close contacts with the G1 peptide in red. The backbone of G1 peptide is colored green with the sidechains of all residues within the PPRPPKP motif depicted in blue. (b) Ribbon representation of the cSH3 domain bound to G2 peptide. The β-strands in the cSH3 domain are shown in yellow with loops depicted in gray and the sidechains of key residues involved in making close contacts with the G2 peptide in red. The backbone of G2 peptide is colored green with the sidechains of conserved residues within the PXVXRXLKPXR motif depicted in blue.

Article Snippet: 12-mer wildtype and mutant peptides spanning G1, G2, G3 and G4 sites within human Gab1 were commercially obtained from GenScript Corporation.

Techniques: Derivative Assay