Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Schematic for in silico pK a prediction method for proteins with solved structures (see text and methods for details). Briefly, all available structures in the protein database are curated, electrostatic properties are calculated using PROPKA, results are filtered for ionizable residues with physiologically relevant predicted pK a values, and data are visualized in a 3D structure or a 2D residue interaction network. (b) Crystal structure of SHP2 shown in cartoon and surface format (PDB ID:2SHP). Protein tyrosine phosphatase (PTP) domain colored in grey, SH2 domains colored in yellow. (c) Structure of SHP2 (PDB ID:2SHP) with protein tyrosine phosphatase (PTP) domain in grey and SH2 domains in yellow. Residues identified through in silico ionizable network prediction pipeline shown in spheres. Residues with predicted pK a shifts (cyan) cluster with ionizable interactors (magenta) across the phosphatase-SH2 domain interaction interface of SHP2. (d) Table of predicted pK a s for cyan residues identified using in silico ionizable network prediction pipeline on 47 SHP2 structures (mean ± SD). (e) Residue interaction network of residues with predicted pK a shifts (cyan) and their ionizable interactors (magenta). Length of edges reflect the strength of the coulombic interaction, with stronger coulombic interactions having shorter edge lengths (f) Zoom of SHP2 structure at the PTP-SH2 interaction interface. Networked residues from a and b are shown in stick. Residues with predicted pK a shifts in cyan and ionizing interactors in magenta.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: In Silico, Residue

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Wild-type (WT) SHP2 in vitro phosphatase activity curves with increasing concentrations of generic substrate p-Nitrophenyl Phosphate (PNPP) at buffer pH ranging from 6.1 to 8.0. (mean ± SEM; N=3 from ≥2 different protein preparations) (b) Double mutant (H116A/E252A) SHP2 in vitro phosphatase activity, assays performed as in A. (mean ± SEM; N=3 from ≥2 different protein preparations) (c) Plot of k cat vs. pH for WT and double mutant (H116A/E252A) SHP2 activity. Calculated from activity curves in a and b. (mean ± SEM) (d) Single-mutant H116A-SHP2 in vitro phosphatase activity, assays performed as in a. (mean ± SEM; N=3 from ≥2 different protein preparations) (e) Single-mutant E252A-SHP2 in vitro phosphatase activity, assays performed as in a. (mean ± SEM; N=3, from ≥2 different protein preparations) (f) Plot of K cat vs. pH for WT and double mutant (H116A/E252A) SHP2 activity. Calculated from activity curves in a, d and e. (mean ± SEM) (g) Proposed pH-sensing mechanism where SH2 domain (yellow) binding to catalytic domain (grey) is titratable by pH. (h) CpHMD (see methods for details) was performed on SHP2 at pH values from 4.0-10.0 (see supplemental videos). Shown are overlapping views of SHP2 structures at the start of CpHMD simulation (t = 0 ns) and end of simulation (t = 8 ns) for pH values 4.5 (pink), 5.5 (purple), 6.5 (blue) and 8.5 (yellow).

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: In Vitro, Activity Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) pHi measurements of MCF10A cells. Cells were treated with 25 μM EIPA + 30 μM S0858 for 1 hour to lower pH to 7.10. To raise pHi, cells were treated with 30 mM ammonium chloride for 1 hour to raise pH to 8.00. Untreated cells had a pHi of 7.45. Scatter plot shows (median ± interquartile range, N =10) (b) Representative immunoblots of SHP2, Gab1, and phospho-SHP2 (pY542) from SHP2 immune complexes (SHP2 IP) or whole cell lysates (Cell Lysate) isolated from MCF10A cells prepared as in A. (c) Quantification of replicate data collected as in B. Data was normalized to control in each biological replicate. Scatter plot shows (mean ± SEM, N=4). I (d) Quantification of Co-IP of SHP2 shown in b. Immunoblot intensities in the treatment conditions were normalized to control in each biological replicate. Scatter plot shows (mean ± SEM, N=7). (e) Representative images of MCF10A cells expressing the SHP2 activity reporter (Grb2 TagBFP) pseudocolored on an intensiometric scale. Images show cells prior to manipulating pHi with nigericin buffer (Pre Nigericin) (see methods for details), 50s - after manipulating pHi, and 900s after manipulating pHi. Scale bars: 25μm (f) Quantification of images as in E. Membrane intensity of SHP2 activity reporter was photobleach-corrected and then normalized to initial intensity over time. Line trace shows from single-cell data (mean ± SEM) (6.7 pH, n=30, 7.4 pH, n=30, 7.8 pH, n=25, control, n=28) collected across N=3 biological replicates. (g) Quantification of endpoint membrane intensities of single cells collected as described in f. Scatter plot shows (median ± interquartile range, N = 5) (h) Representative immunoblot of lysates prepared from MCF10A cells expressing either WT SHP2 or H116A/E252A SHP2 and treated as described in a. Immunoblots show total and pY542-SHP2 under low, control, and high pHi conditions. (i) Quantification of replicate data collected as in h. Scatter plots show (mean ± SEM, N=3). Intensities were normalized to the corresponding control condition. For a and g significance was determined using the Kruskal-Wallis test. For c, d, and i significance was determined using a ratio paired t-test to compare between treatment conditions and a one-sample t-test to compare to control. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: Western Blot, Isolation, Control, Co-Immunoprecipitation Assay, Expressing, Activity Assay, Membrane

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Schematic of pH-driven activation and inhibition of SHP2. At low pH, the SH2 domain is unbound and SHP2 becomes signaling active with increased phosphorylation of Y542, increased GAB1 binding, and increased Grb2 recruitment. (b) Schematic of pH-driven activation and inhibition of Src. At low pH, the SH2 domain is unbound and c-Src becomes signaling active with increased phosphorylation of Y416, decreased phosphorylation of Y527, and increased membrane recruitment.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: Activation Assay, Inhibition, Phospho-proteomics, Binding Assay, Membrane

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Identification of FHL2 as a novel interacting protein of IER3. ( a ) Yeast growth was demonstrated in colonies expressing both FHL2 and IER3 fused to the GAL4 DNA activation and binding domains, respectively. ( b , c ) 293T cells were co-transfected with Myc-FHL2 and FLAG-IER3 ( b ) or HA-FHL2 and FLAG-IER3 ( c ). After 24 h of incubation, cell lysates were prepared and immunoprecipitated with anti-FLAG ( b ) or anti-HA ( c ) antibodies. Immunoblot analyses were performed using the indicated antibodies. ( d ) The interaction between endogenous FHL2 and IER3 proteins was determined in HeLa cells after immunoprecipitation with control IgG or an anti-IER3 antibody. For all immunoblot images presented throughout this manuscript, the membrane was cut into pieces according to the estimated molecular weight of the proteins of interest and probed with the indicated antibodies. All cropped blots were run under the same experimental conditions. ( e ) Intracellular co-localization of endogenous FHL2 and IER3 proteins in HeLa cells was assessed by fluorescence confocal microscopy.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Expressing, Activation Assay, Binding Assay, Transfection, Incubation, Immunoprecipitation, Western Blot, Molecular Weight, Fluorescence, Confocal Microscopy

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Mapping of the binding regions for the interaction between FHL2 and IER3. ( a ) Structures of the plasmids encoding HA-tagged full-length and truncated mutants of FHL2 are illustrated. ( b ) HeLa cells were co-transfected with each HA-tagged FHL2 construct and GST-IER3. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated with GST beads. Arrows indicate expected positions of FHL2 proteins. GAPDH was included as a loading control. ( c ) Structures of GST-tagged full-length and deletion mutants of IER3 are illustrated. ( d ) Immunoprecipitation was performed as described in b following transfection with GST-IER3 constructs and Myc-FHL2 into HeLa cells. Arrows indicate IER3 proteins. ( e ) Transfection and immunoprecipitation were performed as described in b with indicated DNA constructs. Arrows indicate expected sizes of FHL2 mutants.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Binding Assay, Transfection, Construct, Immunoprecipitation

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Regulation of IER3 stability by FHL2 via the promotion of ubiquitination-mediated proteasomal degradation. ( a ) HeLa cells were transfected with FLAG-IER3 in the presence or absence of HA-FHL2. The pEGFP plasmid (300 ng) was co-transfected as an internal control of transfection efficiency. Twenty-four hours after transfection, cell lysates were prepared and subjected to SDS–polyacrylamide gel electrophoresis. The IER3 level was determined by western blot analysis. ( b ) Immunoblot (IB) results from HeLa cells after knockdown of FHL2 by FHL2 siRNA (100 and 200 nM) transfection are shown. ( c ) Twenty-one hours after transfection of HeLa cells with FHL2 siRNA, the cells were incubated with cycloheximide (CHX; 100 μg/ml), harvested at the indicated time points and analyzed by IB. Quantitative analysis of IER3 levels from three independent experiments is presented (lower panel). Asterisks indicate statistically significant differences ( P <0.05). ( d) HeLa cells were co-transfected with FLAG-IER3 and HA-FHL2 or the pCMV-HA empty vector. Twelve hours after transfection, the cells were treated with dimethylsulphoxide (0.1%), MG132 (50 μM) or chloroquine (50 μM) for 12 h. The cell lysates were subjected to IB analysis. ( e , f ) HeLa cells were transfected with FLAG-IER3 and HA-ubiquitin together with HA-FHL2 or HA-FHL2ΔC2 ( e ), or FHL2 siRNA ( f ). Twelve hours after transfection, the cells were incubated with MG132 (50 μM) for 12 h followed by immunoprecipitation (IP) and IB analysis. Arrows indicate the expected position of mono-, di- and tri-ubiquitinated IER3. ( g ) HeLa cells were transfected with siRNA for FHL2. Twelve hours after transfection, the cells were incubated with MG132 (50 μM) for 12 h followed by IP and IB analysis.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Transfection, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Western Blot, Incubation, Immunoprecipitation

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Identification of IER3 lysine 60 as the ubiquitin acceptor site. ( a ) The alignment of amino-acid sequences of mammalian IER3 proteins encompassing two lysines, K60 and K84 (red), is shown. ( b ) The stability of the GST-tagged IER3 wild-type (WT) and substitution mutants was determined after transfection of HeLa cells with the respective plasmids. Twelve hours after transfection, the cells were incubated with MG132 (50 μM) for 12 h, and cell lysates were analyzed by western blot analysis. ( c , d ) Changes in the protein stability of IER3 WT and mutants in response to FHL2 knockdown ( c ) or overexpression ( d ) were assessed by western blot analysis. A green fluorescent protein (GFP)-expressing plasmid was included as an inner control of transfection efficiency. ( e ) An in vivo ubiquitination assay was performed after transfection of HeLa cells with the indicated plasmids followed by immunoprecipitation.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Transfection, Incubation, Western Blot, Over Expression, Expressing, Plasmid Preparation, In Vivo, Ubiquitin Assay, Immunoprecipitation

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Regulation of MDM2-mediated degradation of IER3 by FHL2. ( a , b ) Changes in IER3 levels were determined by western blot analysis in HeLa cells after transfection with the indicated plasmids and siRNAs (200 nM). ( c ) The direct interaction between FHL2 and MDM2 was assessed by an in vitro immunoprecipitation (IP) after incubation of recombinant FHL2 (1 μg) and GST-MDM2 (0.5 μg) proteins. ( d ) The HA-tagged WT and mutant FHL2 constructs shown in were co-transfected with MDM2 into HeLa cells and their binding capacities were assessed by IP. The arrows indicate the expected positions of FHL2 proteins. ( e ) The GST-tagged WT and mutant MDM2 constructs presented in were co-transfected with Myc-FHL2 into HeLa cells and their binding capacities were assessed by IP. The arrows indicate the expected positions of MDM2 proteins. ( f , g ) The effect of FHL2 expression on the association of IER3 and MDM2 was determined after transfection of HeLa cells with the indicated plasmids and siRNA followed by IP and western blot analysis. The relative interaction ratios are graphically presented from the results of three independent experiments ( P <0.05). ( h ) The interaction between endogenous IER3 with endogenous FHL2 and MDM2 proteins were determined in HeLa cells after IP with a BS3-crosslinked anti-MDM2 antibody or control IgG. ( i ) Formation of a ternary complex in vitro was determined after incubation of 1 μg of recombinant proteins (FHL2, MDM2 and GST-IER3) followed by IP and western blot analysis. ( j ) A direct effect of FHL2 on the MDM2-mediated IER3 ubiquitination was assessed in an in vitro ubiquitination assay. Purified recombinant FHL2 (1 μg) and GST-IER3 (1 μg) proteins were incubated in Ub buffer containing MDM2 protein (0.3 μg). IB, immunoblot.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Western Blot, Transfection, In Vitro, Immunoprecipitation, Incubation, Recombinant, Mutagenesis, Construct, Binding Assay, Expressing, Ubiquitin Assay, Purification

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Inhibition of IER3-induced cell cycle arrest by FHL2 and MDM2. Effects of FHL2 and IER3 on the cell cycle regulation were assessed after transfecting HeLa cells with IER3- or FHL2-encoding plasmids ( a ) and specific siRNA for IER3 ( b ). Twenty-four hours post transfection, the cells were harvested and analyzed by flow cytometry. Representative DNA content histograms are presented (left panel). Cell populations in the S phase were graphically presented as % of control. Asterisks indicate statistically significant differences from control values determined after transfection with an empty vector ( P <0.05). ( c ) HeLa cells were transfected with the IER3 plasmid with or without siRNAs specific for MDM2 or FHL2 for 24 h and cell cycle phases were analyzed (top panel). Efficient silencing of FHL2 and MDM2 using specific siRNAs (200 nM) was confirmed by western blot analysis and is presented in the bottom panel. Different letters denote statistically significant differences ( P <0.05). Data are presented as the mean±s.e.m. from three independent experiments performed in duplicate. ( d , e ) MDM2 +/+ / p53 −/− and MDM2 −/− / p53 −/− MEF cells were transfected with FLAG-IER3 in the presence or absence of HA-FHL2. Twenty-four hours after transfection, immunoblotting ( d ) and 5′-bromo-2′-deoxyuridine cell proliferation assays ( e ) were performed. Different letters denote statistically significant differences ( P <0.01). Data are presented as the mean±s.e.m. of three independent experiments performed in triplicate. ( f ) HeLa cells were transfected with siRNAs for HPV18 E6 or E7 (200 nM). Twenty-four hours after transfection, cell lysates were prepared and immunoblotted with the indicated antibodies. ( g ) HeLa cells were transfected with siRNAs for HPV18 E6 and/or MDM2 (200 nM) for 24 h. The cell lysates were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Inhibition, Transfection, Flow Cytometry, Plasmid Preparation, Western Blot

Journal: Oncogene

Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells

doi: 10.1038/onc.2016.54

Figure Lengend Snippet: Upregulated FHL2 and downregulated IER3 in squamous epithelium of cervical cancer tissues. ( a ) Scatter histograms of the score determined by immunohistochemical staining of FHL2, IER3 and MDM2 are presented. Statistical differences in immunostaining analysis between cervical carcinoma (CA) and non-cancer (NC) cervical tissues in the TMA performed by two-tailed t -tests are shown. ( b ) ROC curve analysis of the proportion scores (PS), intensity scores (IS), and total scores (TSs) shows similar AUC (area under the curve) values. TSs were calculated as the sum of the PS in the range of 0–5 and the IS within the range of 0–3. ( c ) A proposed molecular mechanism involved in the regulation of IER3 degradation by FHL2 and MDM2 is presented.

Article Snippet: The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA).

Techniques: Immunohistochemical staining, Staining, Immunostaining, Two Tailed Test