Journal: International journal of molecular sciences

Article Title: Protein Tyrosine Phosphatase Non-Receptor 11 ( PTPN11 /Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

doi: 10.3390/ijms241310545

Figure Lengend Snippet: Figure 1. PTPN11 mutation occurrence and co-occurrence alongside other mutations in both lung adenocarcinomas and squamous cell carcinomas. PTPN11 mutation occurrence rate across the genotyped tumour tissue of NSCLC patients (n = 356) and TCGA data (n = 586) (A). Oncoprint shows the gene alterations in each individual with PTPN11-mutated NSCLC (n = 37), focusing on known cancer-related genes. Each box represents a patient; genes and corresponding alteration frequencies are listed (B). The type of PTPN11 mutation occurring across both adenocarcinomas (LUAD) and squamous cell carcinoma cohorts (LUSC) is displayed (n = 37) (C).

Article Snippet: The constructs expressing PTPN11 wild type (WT), E76A, and C459S (Ben Neel, Addgene plasmids 8329, 8331, and 8382, respectively) were obtained.

Techniques: Mutagenesis

Journal: International journal of molecular sciences

Article Title: Protein Tyrosine Phosphatase Non-Receptor 11 ( PTPN11 /Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

doi: 10.3390/ijms241310545

Figure Lengend Snippet: Figure 2. PTPN11 mutations promote IL-3-independent survival of Ba/F3 cells. The stable expression of E76A and A72D PTPN11 mutations promoted IL-3 independent survival of Ba/F3 cells. Ba/F3 cells transduced with indicated vectors (EV = empty vector, pBabe) were plated in the absence of IL-3. Viable cells were determined at 0 h, 24 h, 48 h, 96 h, and 120 h. Results are representative of three independent experiments. Data are represented as mean ± s.e.m. **** p < 0.0001.

Article Snippet: The constructs expressing PTPN11 wild type (WT), E76A, and C459S (Ben Neel, Addgene plasmids 8329, 8331, and 8382, respectively) were obtained.

Techniques: Expressing, Transduction, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: Protein Tyrosine Phosphatase Non-Receptor 11 ( PTPN11 /Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

doi: 10.3390/ijms241310545

Figure Lengend Snippet: Figure 3. PTPN11 mutations result in elevated SHP2-phosphatse activity and activate MAPK and PI3K pathway signalling. Serum-starved cells treated with 100 ng/mL EGF for 5 min were lysed, and SHP2 was immunoprecipitated from whole cell lysates. Immunoprecipitate was used to de- termine phosphatase activity, as described in the materials and methods. (A) SHP2-phosphatse activity in H661 (PTPN11-mutated) compared to H1703 (PTPN11-WT), Calu-3 (PTPN11-WT), and H157 (PTPN11-WT, KRAS-mutated). Shp2-phosphatase activity in H1701 (B) and H1299 (C) cells transduced with indicated PTPN11 mutations. Data are represented as mean ± s.e.m. (n = 3). (D) NCI-H1703 and NCI-H157 cells were transduced with wildtype or mutated PTPN11: a serum starved and stimulated with an epidermal growth factor (100 ng/mL) for 5 min. p-ERK1/2—phospho- ERK 1/2; t-ERK1/2—total ERK 1/2; p-AKT ser 473—phospho-AKT (phosphorylated at serine 473); t-AKT—total AKT. Blots are representative of 3 independent experiments. (NS = No Significance, * p < 0.05, ** p < 0.01, *** p <0.001)

Article Snippet: The constructs expressing PTPN11 wild type (WT), E76A, and C459S (Ben Neel, Addgene plasmids 8329, 8331, and 8382, respectively) were obtained.

Techniques: Activity Assay, Immunoprecipitation, Transduction

Journal: International journal of molecular sciences

Article Title: Protein Tyrosine Phosphatase Non-Receptor 11 ( PTPN11 /Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

doi: 10.3390/ijms241310545

Figure Lengend Snippet: Figure 4. PTPN11/Shp2 inactivation with the PTPN11/Shp2 phosphatase mutation C459S. NCI-H157, NCI-H1703, and NCI-H661 cells were transduced with PTPN11 C459S: serum starved and stimulated with an epidermal growth factor (100 ng/mL) for 5 min. Parental cells were transfected with an empty vector. Phosphorylated-ERK 1/2 (pERK); total ERK 1/2 (tERK); Phosphorylated AKT (phosphory- lated at serine 473) (pAKT); total AKT (tAKT). Blots are representative of 3 independent experiments.

Article Snippet: The constructs expressing PTPN11 wild type (WT), E76A, and C459S (Ben Neel, Addgene plasmids 8329, 8331, and 8382, respectively) were obtained.

Techniques: Mutagenesis, Transduction, Transfection, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: Protein Tyrosine Phosphatase Non-Receptor 11 ( PTPN11 /Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

doi: 10.3390/ijms241310545

Figure Lengend Snippet: Figure 5. SHP2 inhibitor (SHPi) improves the response to MAPK and PI3K pathway targeting therapies. H661 and H1703 cells plated and after 24 h were treated with SHPi (10 µM) daily or Cop (20 nM) or Ref (10 µM) alone or in combination with SHPi. 72 h following treatment, an Alamar Blue cell viability assay was performed (A). Data (n = 3 independent experiments) are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA correcting for multiple comparisons using Tukey’s test and reporting adjusted p values. (* p < 0.05, ** p < 0.01).

Article Snippet: The constructs expressing PTPN11 wild type (WT), E76A, and C459S (Ben Neel, Addgene plasmids 8329, 8331, and 8382, respectively) were obtained.

Techniques: Viability Assay

Journal: International journal of molecular sciences

Article Title: Protein Tyrosine Phosphatase Non-Receptor 11 ( PTPN11 /Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

doi: 10.3390/ijms241310545

Figure Lengend Snippet: Figure 7. PTPN11 and PI3K targeting treatments do not alter tumour formation or invasion in a chick embryo xenograft model. 2 × 106 H661 cells were implanted into the CAM according to the assay schedule (A) on day 7 of embryonic development. Following 72 h of tumour establishment, developing tumours were treated in situ. SHPi (10 µM) treatments were added daily, and Cop (20 nM) treatments were added once on day 10. On day 14, tumour visibility was noted as either visible (VT) or non-visible (NVT). Statistical significance was determined using Fisher’s exact test, and no statistical significance was found (B) On day 14, xenografts were excised with the silicon ring, formalin-fixed, and stained for H&E (C,D). Areas of tumour were denoted by black arrows, and CAM areas were demonstrated by red arrows and matrigel was denoted by grey arrows. Images were collected an EVOS m5000 microscope with EVOS imaging software at 4× and 20× magnification (D).

Article Snippet: The constructs expressing PTPN11 wild type (WT), E76A, and C459S (Ben Neel, Addgene plasmids 8329, 8331, and 8382, respectively) were obtained.

Techniques: In Situ, Staining, Microscopy, Imaging, Software

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Inhibition of FGF signaling did not activate Src or FAK. BAEC were transduced with Ad-GFP or Ad-FGFR1DN. Cells in normal growth medium were lysed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. B , C , D , Quantitative analysis of pY416 Src (B), pY527 Src (C), pY397 Fak (D) shown in Fig. 3A. The value of NT, standardized with β-tubulin, was designated as 1. (n = 3). E , F . Reduced SHP2 expression in cells lacking FGF signaling. Western analysis using BAEC total cell lysates left untreated or transduced with either Ad-GFP or Ad-FGFR1DN. G , Quantitative analysis of SHP2 levels shown in Fig. 3F. The value of Ad-GFP at MOI 37.5, standardized with β-tubulin, was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with Ad-GFP, MOI 37.5). H , I . VE-cadherin-SHP2 interaction was disrupted by FGF signaling inhibition. BAEC were transduced with Ad-GFP or Ad-FGFR1DN. Cells were lysed and immunoprecipitated (IP) with anti-VE-cadherin (H) or anti-SHP2 (I) and subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. NT denotes no transduction. J . Catalytically inactive, dominant-negative SHP2 increased Y658 VE-cadherin phosphorylation. BAEC were transduced with lentivirus wild-typeSHP2 (WT) or dominant-negative-SHP2 (C/S). Cells were lysed and total cell lystes were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Inhibition, Transduction, SDS Page, Western Blot, Expressing, Immunoprecipitation, Dominant Negative Mutation, Phospho-proteomics

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. SHP2 mRNA levels were not decreased in endothelial cells lacking FGF signaling. Quantitative RT-PCR analysis of total RNA isolated from BAEC. Total RNA was isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN. SHP2 mRNA levels were measured with real-time PCR and normalized to GAPDH expression (Mean ± SD, * P <0.05, by t-test compared with NT). NT denotes no transduction. B . Western blotting of total cell lysates isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN and treated with 10 µg/ml cycloheximide for up to 36 hours. C . Quantitative analysis of SHP2 Western analysis described in B. The value at time point 0 was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with Ad-GFP). D . SHP2 is degraded via the lysosomal pathway in the absence of FGF signaling. Confluent BAEC transduced with either Ad-GFP or Ad-FGFR1DN were treated with 1 µM MG132, 20 µM lactacystin, 20 µM chloroquine or 25 mM NH 4 Cl for 24 hr. Total cell lysates were analyzed by Western blot. E . Quantitative analysis of SHP2 expression shown in Fig. 2D. The value of Ad-GFP control (DMSO) treatment, standardized with β-tubulin, was designated as 1. (n = 3, Mean ± SD, * P <0.05, by t-test compared with Ad-FGFR1DN control).

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Quantitative RT-PCR, Isolation, Transduction, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Increased phosphorylation of VE-cadherin Y658 in cells lacking FGF signaling was restored to the basal level by SHP2 overexpression. BAEC were transduced with Ad-FGFR1DN and Ad-GFP or Ad-SHP2. Cells were lysed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. B . Quantitative analysis of SHP2 shown in Fig. 5A. The value of NT, standardized with β-tubulin, was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with NT, ** P <0.05, by t-test compared with Ad-FGFR1DN+Ad-GFP). C . SHP2 overexpression restored p120-catenin/VE-cadherin association. BAEC were transduced with Ad-FGFR1DN and Ad-GFP or Ad-SHP2. Total cell lysates were isolated and immunoprecipitated with VE-cadherin antibody. Immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies using the same membrane after stripping and reprobing. D , E , Immunostaining of quiescent and fully confluent BAEC transduced with Ad-FGFR1DN (D), or Ad-SHP2 and Ad-FGFR1DN (E). Cells were stained for VE-cadherin (green), SHP2 (red), and HA (FGFR1DN, blue). Arrows indicate gap formations between cells. F . Quantitative analysis of immunostaining evaluating the gap formation. Percent of gap area in each image was calculated using NIH Image J software using 6 different images. Data shown as mean ± SD *: P <0.05 by t-test. Scale Bars: 10 µm. G . SHP2 overexpression rescued the FGFR1DN effect on endothelial permeability. BAEC were transduced with Ad-SHP2 and Ad-FGFR1DN, and endothelial monolayer permeability was evaluated with the ECIS system. Transendothelial resistance was measured every 5 minutes for 17 hours after the onset of adenoviral transduction. NT denotes no transduction.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Phospho-proteomics, Over Expression, Transduction, SDS Page, Western Blot, Isolation, Immunoprecipitation, Membrane, Stripping Membranes, Immunostaining, Staining, Software, Permeability

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Under normal circumstances, SHP2 is associated with VE-cadherin and dephosphorylates Y658 site of VE-cadherin, which results in p120-catenin coupling, enhancing VE-cadherin retention at adherens junctions. FGF signaling is required for VE-cadherin-SHP2 interaction. B . In cells expressing the FGFR1DN construct, SHP2 is downregulated and is not able to associate with VE-cadherin due to the lack of FGF signaling, which increases phosphorylation level of VE-cadherin Y658, leading to decoupling of p120-catenin; therefore, VE-cadherin stability at cell-cell junctions is impaired.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Expressing, Construct, Phospho-proteomics

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Domain architecture diagram of SHP2. SHP2 consists of two SH2 domains (yellow and pink) and a phosphatase domain (green). Relevant mutations and the catalytic cysteine (C459) are indicated. ( B ) SHP2 is kept in its auto-inhibited state by interactions between the N-SH2 and PTP domain (PDB: 4DGP). In its active state, the N-SH2 domain is pulled away, and the catalytic cysteine is accessible. Although the structure of SHP2 E76K (PDB: 6CRF) is used to represent the active state, multiple active states likely exist. ( C ) SHP2 is activated by upstream stimuli. The SH2 domains bind to tyrosinephosphorylated upstream proteins, such as transmembrane receptors, inducing a conformational change that activates SHP2. ( D ) Disease-associated mutations cluster largely, but not exclusively, on the interdomain interface between the N-SH2 and the PTP domain (PDB: 4DGP). Highlighted unlabeled mutation sites include: N58, G60, Y62, E69, F71, A72, E76, Q79, D106, E110, Q256, G268, Y279, I282, F285, N308, I309, T411, A461, G464, T468, R498, R501, M504, Q510. ( E ) Mutations in or near the N-SH2 binding pocket (PDB: 6ROY). T42 is engaging the phosphotyrosine of the phosphopeptide ligand, whereas L43 is facing into the SH2 domain core. T52 is near the residues surrounding the phosphotyrosine. ( F ) Mutations in or near the C-SH2 binding pocket (PDB: 6R5G). R138 is engaged with the phosphotyrosine of the phosphopeptide ligand, whereas E139 is facing away from the binding pocket.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Mutagenesis, Binding Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Measured binding affinities of N-SH2 WT against peptides derived from various known SHP2 interactors. ( B ) Fold-change in K D for N-SH2 T42A compared to N-SH2 WT , for each of the peptides shown in panel (A). ( C ) Same as (B), but for N-SH2 L43F . ( D ) Same as (B), but for N-SH2 T52S . Source data can be found in Table S2.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Hydrogen bonding of Thr 42 in SHP2 N-SH2 WT to the phosphoryl group of phosphopeptide ligands, as seen in several crystal structures (PDB codes: 6ROY, 1AYA, 1AYB, 3TL0, 5DF6, 5X94, and 5X7B). ( B ) Structure of N-SH2 WT bound to the PD-1 pTyr 223 (ITIM) peptide at the end of a 1 μs MD simulation, highlighting a hydrogen bond network and other key interactions around the phosphotyrosine residue. ( C ) Structure of N-SH2 T42A bound to the PD-1 pTyr 223 (ITIM) peptide at the end of a 1 μs MD simulation, highlighting a distinct hydrogen bond network around the phosphotyrosine residues, relative to that seen for N-SH2 WT . ( D ) Overlay of the states shown in panels B and C, highlighting a change in position for the phosphotyrosine residue and peptide main chain upon T42A mutation. The N-SH2 WT state is in yellow with a dark-gray ligand. The N-SH2 T42A state is in light gray, with a light gray ligand. ( E ) Distribution of distances between the Lys 55 Nζ atom and the phosphotyrosine phosphorus atοm in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( F ) Distribution of distances between the Lys 55 Nζ atom and the +2 Glu Cδ atom in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( G ) An ion pair between Lys 55 and the +2 Glu residue (Glu 225) in the PD-1 pTyr 223 (ITIM) peptide, frequently observed in N-SH2 T42A simulations. ( H ) Effects of the T42A mutation in the context of the K55R mutation. The enhancement in binding affinity by the T42A mutation is attenuated by the K55R mutation for some peptides (CagA-D, PD-1 pTyr 223, and MILR1 pTyr 338) but not others (IRS1 pTyr 1179 and Imhof-9).

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Phospho-proteomics, Residue, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) SHP2 activation is measured by incubation with phosphopeptide ligands, followed by monitoring dephosphorylation of the small-molecule substrate DiFMUP to generate fluorescent DiFMU. ( B ) Representative activation curves for SHP2 WT , highlighting peptide-dependent changes in EC 50 and amplitude. ( C ) Correlation between the EC 50 of SHP2 WT activation by phosphopeptides and the K D of those phosphopeptides for the N-SH2 WT domain. ( D ) Correlation between activation EC 50 values for SHP2 WT and SHP2 R 138 Q , which has weakened C-SH2 binding capacity. ( E ) Comparison of SHP2 WT and SHP2 T42A activation curves for the PD-1 pTyr 248 peptide, highlighting a significant impact on both EC 50 and amplitude. ( F ) Comparison of SHP2 WT and SHP2 T42A activation curves for the Imhof-9 peptide, highlighting a minor change in EC 50 and amplitude. ( G ) Bubble plot juxtaposing the EC 50 values for activation of SHP2 WT and SHP2 T42A by nine peptides, alongside the fold-change in K D for binding of those peptides to N-SH2 WT vs N-SH2 T42A . The dotted line indicates where EC 50 values would be equivalent for SHP2 WT and SHP2 T42A . The graph shows that peptides with a large fold-change in binding affinity (larger bubble) have a large fold-change in EC 50 values for SHP2 T42A over SHP2 WT (distance from dotted line). All EC 50 values can be found in Table S5.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Activation Assay, Incubation, Phospho-proteomics, De-Phosphorylation Assay, Binding Assay, Comparison

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires interaction specificity and enhances signaling

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Schematic diagram depicting the co-immunoprecipitation (co-IP) experiments with SHP2 and either Gab1, Gab2, or PD-1 in HEK293 cells. The interactor proteins are phosphorylated by a hyperactive form of c-Src kinase. SHP2 co-immunoprecipitation experiments with ( B ) Gab1, ( C ) Gab2, and ( D ) PD-1, demonstrating that SHP2 T42A binds tighter to these phosphoproteins than SHP2 WT . In each case, SHP2 was immunoprecipitated via its myc-tag. Co-immunoprecipitation of the interacting protein was detected using an α-FLAG antibody for Gab1/Gab2 and a PD-1-specific antibody for PD-1. For PD-1, the experiment was also conducted by immunoprecipitating PD-1 and detecting co-immunoprecipitation of SHP2 using an α-myc antibody. ( E ) Schematic depiction of EGF stimulation and phospho-Erk signaling experiments in the presence of co-expressed SHP2 and either Gab1 or Gab2. ( F ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab1 and either SHP2 WT or SHP2 T42A . ( G ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab2 and either SHP2 WT or SHP2 T42A . For panels (F) and (G), the numbers below the blots indicate phospho-Erk levels relative to the 0 minute sample with SHP2 WT .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison, Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC)

doi: 10.1152/ajpgi.00011.2017

Figure Lengend Snippet: Role of SHP2 PTPase in modulating SERT phosphorylation. SHP2 PTPase constructs [wild-type (Wt), constitutively active, and dominant negative (-ve)] were overexpressed in Caco-2 cells by Amaxa electroporation. SERT was immunoprecipitated from whole cell lysates and probed with p-tyrosine antibody to check the phosphorylation levels of SERT at tyrosine residues. The blot was also probed with SHP2 PTPase antibody to show that the total levels of the phosphatase remained unchanged. A: representative Western blot for phosphorylation levels of SERT at tyrosine residues and SHP2 PTPase levels in total lysates. B: densitometric analysis for phosphorylated SERT levels. Results are expressed as p-tyrosine/total SERT. Values represent means ± SE of 3 different experiments. ***P < 0.005 vs. control with one-way ANOVA.

Article Snippet: SHP2 PTPase constructs [wild-type (Wt), constitutively active, and dominant negative (-ve)] were overexpressed in Caco-2 cells by Amaxa electroporation.

Techniques: Phospho-proteomics, Construct, Dominant Negative Mutation, Electroporation, Immunoprecipitation, Western Blot, Control

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC)

doi: 10.1152/ajpgi.00011.2017

Figure Lengend Snippet: Constitutively active construct of SHP2 PTPase inhibits SERT function. Caco-2 cells were transiently transfected with the SHP2 PTPase constructs (wild-type, constitutively active, and dominant negative) by Amaxa electroporation. SERT function was measured 24 h post-transfection as [3H]5-HT uptake (control values in picomoles per milligram of protein per 5 min). *P < 0.05 vs. control, n = 3, by one-way ANOVA.

Article Snippet: SHP2 PTPase constructs [wild-type (Wt), constitutively active, and dominant negative (-ve)] were overexpressed in Caco-2 cells by Amaxa electroporation.

Techniques: Construct, Transfection, Dominant Negative Mutation, Electroporation, Control

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC)

doi: 10.1152/ajpgi.00011.2017

Figure Lengend Snippet: Interaction of SERT with SHP2 PTPase in Caco-2 cells. Caco-2 cells were transiently transfected with wild-type (w+), constitutively active (c+), and dominant-negative (e−) constructs of SHP2 PTPase by Amaxa electroporation. SERT was immunoprecipitated from the whole cell lysates. The samples were analyzed by 7.5% SDS-PAGE, followed by transfer of proteins to nitrocellulose, and probed with SHP2 antibody. The blots were stripped and reprobed with the SERT antibody to show equal loading. A: representative Western blot showing the association of SHP2 with SERT in different constructs of SHP2 PTPase. B: densitometric analysis for SHP2 PTPase with total levels of SERT. Values represent means ± SE of 3 different experiments. **P < 0.05 vs. control by one-way ANOVA test.

Article Snippet: SHP2 PTPase constructs [wild-type (Wt), constitutively active, and dominant negative (-ve)] were overexpressed in Caco-2 cells by Amaxa electroporation.

Techniques: Transfection, Dominant Negative Mutation, Construct, Electroporation, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Domain architecture diagram of SHP2. Relevant mutations and the catalytic cysteine (Cys 459 ) are indicated. ( B ) SHP2 is kept in its auto-inhibited state by interactions between the N-SH2 and PTP domain (PDB: 4DGP). In its active state, the catalytic cysteine is accessible. The structure of SHP2 E76K (PDB: 6CRF) is used to represent the active state. ( C ) The SH2 domains of SHP2 bind to upstream phosphoproteins, such as transmembrane receptors, inducing a conformational change that activates SHP2. ( D ) Disease-associated mutations cluster largely, but not exclusively, on the interdomain interface between the N-SH2 and the PTP domain (PDB: 4DGP). The E76K mutation is a canonical N-SH2/PTP interface mutation. ( E ) Mutations in or near the N-SH2 binding pocket (PDB: 6ROY). ( F ) Mutations in or near the C-SH2 binding pocket (PDB: 6R5G). The well-established specificity-determining regions of the SH2 domains, which dictate +1 to +5 residue preferences, are marked with black dashed lines.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Mutagenesis, Binding Assay, Residue

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Measured binding affinities of N-SH2 WT against peptides derived from various known SHP2 interactors ( B ) Fold-change in K D for N-SH2 T42A compared to N-SH2 WT , for each of the peptides shown in panel (A). ( C ) Same as (B), but for N-SH2 L43F . ( D ) Same as (B), but for N-SH2 T52S . For ( A )-( D ), N = 3–4 independent protein, peptide, and fluorescent peptide titrations. Source data can be found in .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Hydrogen bonding of Thr 42 in SHP2 N-SH2 WT to the phosphoryl group of phosphopeptide ligands in several crystal structures (PDB: 6ROY, 1AYA, 1AYB, 3TL0, 5DF6, 5X94, and 5X7B). ( B ) Representative structure of N-SH2 WT bound to the PD-1 pTyr 223 (ITIM) peptide at the end of one MD simulation. N = 3 simulations of 1 μs each. ( C ) Representative structure of N-SH2 T42A bound to the PD-1 pTyr 223 (ITIM) peptide at the end of one MD simulation. N = 3 simulations of 1 μs each. ( D ) Overlay of the representative states shown in panels B and C. The N-SH2 WT state is in yellow with a dark-gray ligand. The N-SH2 T42A state is in light gray, with a light gray ligand. ( E ) Distribution of distances between the Lys 55 Nζ atom and the phosphotyrosine phosphorus atοm in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( F ) Distribution of distances between the Lys 55 Nζ atom and the +2 Glu Cδ atom in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( G ) An ion pair between Lys 55 and the +2 Glu residue (Glu 225) in the PD-1 pTyr 223 (ITIM) peptide, frequently observed in N-SH2 T42A simulations. ( H ) Peptide-specific effects of the T42A mutation in the presence and absence of the K55R mutation. N = 2–5 independent titrations.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Phospho-proteomics, Residue, Mutagenesis

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) SHP2 activation is measured by incubation with phosphopeptide ligands, followed by monitoring dephosphorylation of the small-molecule substrate DiFMUP to generate fluorescent DiFMU. ( B ) Representative activation curves for SHP2 WT . N = 3–17 independent titrations of protein, peptide, and DiFMUP. ( C ) Correlation between the EC 50 of SHP2 WT activation by phosphopeptides and the K D of those phosphopeptides for the N-SH2 WT domain. For EC 50 values in ( B )-( C ), N = 3 independent titrations of protein, peptide, and DiFMUP.( D ) Correlation between activation EC 50 values for SHP2 WT and SHP2 R138Q . For SHP2 R138Q EC 50 values, N = 3–5 independent titrations of protein, peptide, and DiFMUP. ( E ) Comparison of SHP2 WT and SHP2 T42A activation curves for the PD-1 pTyr248 peptide. N = 3–4 independent titrations of protein, peptide, and DiFMUP. ( F ) Comparison of SHP2 WT and SHP2 T42A activation curves for the Imhof-9 peptide. N = 6–17 independent titrations of protein, peptide, and DiFMUP. ( G ) Bubble plot juxtaposing the EC 50 values for activation of SHP2 WT and SHP2 T42A by nine peptides, alongside the fold-change in K D for binding of those peptides to N-SH2 WT vs N-SH2 T42A . The dotted line indicates where EC 50 for SHP2 WT equals EC 50 for SHP2 T42A . Peptides with a large fold-change in binding affinity (larger bubble) have a large fold-change in EC 50 values for SHP2 T42A over SHP2 WT (distance from dotted line). All EC 50 values can be found in .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Activation Assay, Incubation, Phospho-proteomics, De-Phosphorylation Assay, Comparison, Binding Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Scheme depicting the co-immunoprecipitation experiments with SHP2 and either Gab1, Gab2, or PD-1 in HEK293 cells. SHP2 co-immunoprecipitation results with ( B ) Gab1, ( C ) Gab2, and ( D ) PD-1. For ( B ), ( C ), ( D ), N = 2, 3, and 4 independent cell transfections, respectively. Co-immunoprecipitation of Gab1/Gab2 was detected using an α-FLAG antibody and PD-1 was detected using a PD-1-specific antibody. Co-immunoprecipitation levels of each protein in T42A samples relative to wild-type are normalized for expression level and shown as bar graphs. ( E ) Schematic depiction of EGF stimulation and phospho-Erk signaling experiments in the presence of co-expressed SHP2 and either Gab1 or Gab2. ( F ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab1 and either SHP2 WT or SHP2 T42A . N = 4 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. ( G ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab2 and either SHP2 WT or SHP2 T42A . N = 3 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. For panels (F) and (G), the bar graphs below the blots indicate phospho-Erk levels, normalized to total Erk levels, relative to the highest p-Erk signal in SHP2 WT time course (2 minutes).

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Immunoprecipitation, Transfection, Expressing, Comparison, One-tailed Test