Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: SHP2 inhibits CRC cell proliferation and migration. ( A ) SHP2 knockdown by siSHP2#1 and #2 markedly increased the proliferation of HCT116 and SW480 cells. ( B ) Colony formation assays were conducted to estimate the growth rate of HCT116 and SW480 cells. SHP2 knockdown increased the colony numbers compared with the control group. Representative pictures of colonies (left) and quantification of colony numbers (right) are shown. ( C ) Transwell assay was performed to access the effect of SHP2 on cell migration by siRNA mediated knockdown. Representative pictures of cells (left) and quantification of cell numbers (right) are shown. ( D ) SW480 cells were transfected with pJ-SHP2 plasmid to overexpress SHP2 and the cell proliferation was accessed by MTT assay. ( E ) Colony formation assay was done by overexpression of SHP2 in SW480 cells. SHP2 overexpression decreased the colony numbers compared with the control group. Representative pictures of colonies (left) and quantification of colony numbers (right) are shown. ( F ) Transwell assay was performed to access the effect of SHP2 on cell migration by overexpressing SHP2 in SW480 cells. Representative pictures of cells (left) and quantification of cell numbers (right) are shown. Overexpression of SHP2 in HCT116 and SW480 cells rescued SHP2 knockdown induced cell proliferation ( G ), colony formation ( H ) and cell migration ( I ). ( J and K ) PHPS1 improved HCT116 and SW480 cell proliferation during 3 days in a time- and dose-dependent manner. ( L ) Cells were treated with PHPS1, and colony formation was measured after two weeks by crystal violet staining. PHPS1 increased the number of CRC cell colonies. (M)Transwell assay showing blockade of SHP2 phosphatase activity by PHPS1 improved CRC cell migratory ability. NC, non-silencing control siRNA. Values represent mean ± SEM (n = 3–4), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Migration, Knockdown, Control, Transwell Assay, Transfection, Plasmid Preparation, MTT Assay, Colony Assay, Over Expression, Staining, Activity Assay

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: SHP2 suppressing role in CRC is mediated by STAT3 dephosphorylation. ( A ) STAT3 phosphorylation was increased in CRC cells knocked down for SHP2 by siRNA#1 and #2. ( B ) SHP2 overexpression reduced STAT3 phosphorylation in SW480. ( C ) PHPS1 enhanced the levels of pSTAT3 in 10 mg/L time-dependently. ( D ) Nuclear distribution of pSTAT3 was enhanced after SHP2 knockdown (×400). ( E ) Cryptotanshinone significantly reversed CRC cell proliferation induced by PHPS1. Values represent mean ± SEM (n = 3), **P ≤ 0.01, compared with Vehicle group. ( F ) SW480 was more sensitive to IL-6-induced proliferation after SHP2 knockdown, which was rescued by SHP2 overexpression; meanwhile overexpression SHP2 inhibited IL-6 induced SW480 proliferation. Values represent mean ± SEM (n = 3) # P ≤ 0.05, **P ≤ 0.01, compared with NC + pJ3 group. Whole blots are shown in Supplementary Fig. .

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Over Expression, Knockdown

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Association between the combination of SHP2/nuclear STAT3 and survival in patients with CRC. ( A ) Kaplan–Meier analysis showed that Patients with high SHP2 and low nuclear STAT3 present better DSS (left panel) and DFS (right panel) than patients with low SHP2 and high nuclear STAT3. ( B ) Kaplan-Meier analysis showed that Patients with high SHP2 and low nuclear STAT3 present better DSS (left panel) and DFS (right panel) than patients with low SHP2 or high nuclear STAT3. ( C ) Kaplan-Meier analysis showed that patients with high SHP2 or low nuclear STAT3 present better DFS (right panel) but not DSS (left panel) than patients with low SHP2 and high nuclear STAT3. P values were determined using log-rank test.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques:

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Univariate Cox regression analysis for clinical parameters and the combinations of SHP2 and nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Multivariate Cox regression analysis for clinical parameters and the combinations of high SHP2 and low nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Multivariate Cox regression analysis of DFS for clinical parameters and the combinations of low SHP2 and high nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

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: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 prevents LPS-induced SHP2 oxidation and its inactivation in the protection of AJ integrity. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for 30 min and PTP activity was measured using PTP-specific phosphopeptide as a substrate. B & C. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for the indicated time periods, cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with VE-cadherin or SHP2 antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. D. All the conditions were same as in panel B except that after immunoprecipitation with SHP2 antibodies the immunocomplexes were assayed for SHP2 activity as described in panel A. E. Quiescent HUVECs that were treated with and without LPS (500 ng/ml) in the presence and absence of Allopurinol (50 μM) for 30 min were analyzed for SHP2 activity as described in panel D. F. All the conditions were same as in panel B except the cell extracts were immunoprecipitated with SHP2 antibodies and the immunocomplexes were immunoblotted for Cys sulphonate to measure SHP2 Cys oxidation and the blot was reprobed for total SHP2 levels. G. All the conditions were the same as in panel E except that the cell extracts were analyzed for SHP2 Cys oxidation as described in panel F and the blot was reprobed for total SHP2 levels. H. HUVECs were transiently transfected with empty vector (EV) or Myc-tagged recombinant SHP2 expression vector (WT and C459S mutant), grown to confluence, quiesced, treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and cell extracts were prepared. An equal amount of protein from control and each treatment was immunoprecipitated with Cysteine sulphonate antibody and the immunocomplexes were analyzed by IB for Myc to show SHP2 Cysteine oxidation. An equal amount of protein from control and each treatment was also analyzed by WB for Myc to show the overexpression of SHP2. I. HUVECs that were transfected with WT or mutant SHP2 expression vector and quiesced were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and analyzed for SHP2 activity as described in panel D.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Activity Assay, Phospho-proteomics, Control, Immunoprecipitation, Transfection, Plasmid Preparation, Recombinant, Expressing, Mutagenesis, Over Expression

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Pharmacological inhibition of SHP2 blunts the capacity of RvD1 in the attenuation of LPS-induced Frk activation, α-catenin and VE-cadherin Tyr phosphorylation and AJ disruption. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM), a potent inhibitor of SHP2, for 30 min, cell extracts were prepared and an equal amount of protein from control and each treatment was immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. The same cell extracts were also analyzed for the indicated protein total levels. B. All the conditions were the same as in panel A except that the quiescent HUVEC monolayer after the treatments was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. C & D. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM) for 2 h or the indicated time periods and subjected to dextran flux (C) or TER (D) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Disruption, Control, Immunoprecipitation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Both ALX/FPR2 and GPR32 mediate the protective effects of RvD1 on LPS-induced endothelial AJ disruption and its barrier dysfunction. A. Cell extracts of control and various time periods of LPS (500 ng/ml)-treated HUVECs were analyzed by WB for ALX/FPR2 and GPR32 levels using their specific antibodies. B-D. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) in combination with and without Boc2 (3 μM), ALX/FPR2 inhibitor, for 30 min and XO activity (B), ROS production (C) and SHP2 activity (D) were measured. E. All the conditions were the same as in panel B except that cell extracts were prepared, and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-Cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. F & G. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with and without Boc2 (3 μM) for 2 h or the indicated time periods and subjected to dextran flux (F) and TER (G) assays, respectively. H-J. Quiescent HUVECs were incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and XO activity (H), ROS production (I) and SHP2 activity (J) were measured. K. All the conditions were the same as in panel H except that cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. L. The quiescent HUVEC monolayer that was incubated with either control IgG, GPR32 IgG (10 μg/ml), Boc2 (3 μM) alone or in combination for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. M & N. Quiescent HUVECs monolayer was incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 2 h or the indicated time periods and subjected to dextran flux (M) and TER (N) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control or control IgG; # , p<0.05 vs LPS or con IgG+LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Control, Activity Assay, Immunoprecipitation, Incubation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 attenuates LPS-induced aortic endothelial AJ disruption and hyper-permeability via blocking XO activity and SHP2 inactivation. A. C57BL/6 mice which were kept on chow diet were administered intraperitoneally with RvD1 (10 μg/kg body weight) every 2 days for 3 times before injecting LPS (5 mg/kg body weight) and 24 h later the aortas were isolated, tissue extracts were prepared and an equal amount of protein from each condition was analyzed for XO activity as described in Figure legend 4B. B. All the conditions were the same as in panel A except that tissue extracts containing an equal amount of protein from each condition were immunoprecipitated with Cys sulphonate antibodies and the immunocomplexes were analyzed by IB for SHP2. The same tissue extracts were analyzed by WB for total SHP2 levels. C. All the conditions were the same as in panel A except that tissue extracts were analyzed for SHP2 activity as described in Figure legend 5D. D & E. All the conditions were the same as in panel A except that tissue extracts were immunoprecipitated with pTyr (D) or VE-cadherin (E) antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same tissue extracts were analyzed by WB for the indicated protein total levels. F. All the conditions were same as in panel A except that after isolation the aortas were opened longitudinally, fixed, permeabilized, blocked and co-immunostained for α-catenin and VE-cadherin as described in Figure legend 2E. G. All the conditions were the same as in panel A except that mice were anesthetized and 0.1 ml of 1% Evans Blue (EB) dye was injected into the tail vein. After 20 min, the blood vessels were perfused with PBS through the left ventriculum and the aortas were isolated and photographed. After taking the pictures, the aortas were minced, incubated in formaldehyde solution at 55 °C for 24 h, centrifuged and the optical density of the supernatant was measured at 610 nm in SpectraMax 190 spectrophotometer (Molecular Devices). The aortic endothelial barrier permeability was expressed as ng of EB dye extravasated per mg aorta. The bar graphs represent Mean±SD values of three experiments with 2 animals/group or 5 animals minimum. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Permeability, Blocking Assay, Activity Assay, Isolation, Immunoprecipitation, Injection, Incubation, Spectrophotometry, Control

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: Journal of Neurochemistry

Article Title: PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells 2

doi: 10.1111/j.1471-4159.2007.04782.x

Figure Lengend Snippet: SHP-2 is recruited by cross-linked ICAM-1 and PECAM-1. Panel (a) Cross-linking of ICAM-1 or PECAM-1 was performed on IFN-γ-treated RBE4 cells as described in Material and methods. Immunoprecipitated proteins were eluted with SDS-sample buffer, and resolved on 7.5% SDS-PAGE followed by immunoblotting with anti-phosphotyrosine (4 G10) or anti-ICAM1 or PECAM-1 antibodies or with anti-SHP-2 antibody. Lane a: basal condition; lane b: PECAM-1 cross-linking; lane c: basal condition; lane d: ICAM-1 cross-linking. Panel (b) Sequential cross-linking of ICAM-1 and/or PECAM-1 was performed on IFN-γ-treated mock-transfected HBMECs or stably expressing WT-SHP-2 or Dn-SHP-2 as described in Material and methods. Immunoprecipitated proteins were eluted with SDS-sample buffer, and resolved on 7.5% SDS-PAGE followed by immunoblotting with anti-phosphotyrosine antibody (4 G10) or anti-cortactin antibody. Lanes a, d, g: basal condition; lanes b, e, h: ICAM-1 cross-linking; lanes c, f, i: sequential ICAM-1 and PECAM-1 cross-linking.

Article Snippet: Generation of HBMEC stable transfectants: HBMECs were transfected with either the pcDNA3.1-SHP-2 (human wild type) or pcDNA3.1-DnSHP-2 (Dominant negative) or “empty” plasmid (“mock”) kindly provided by Dr C. Nahmias ( Rivard et al. 1995 ) using the nucleofector system developed by Amaxa Inc (Gaithersburg, MD, USA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Transfection, Stable Transfection, Expressing

Journal: Journal of Neurochemistry

Article Title: PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells 2

doi: 10.1111/j.1471-4159.2007.04782.x

Figure Lengend Snippet: The SHP-2 inhibitor calpeptin mimicks PECAM-1-induced reversal of ICAM-1 signaling. Panel (a) Sequential cross-linking of ICAM-1 and/or PECAM-1 was performed on IFN-γ-treated RBE4 cells pre-incubated with or without the selective SHP-2 inhibitor calpeptin (150 μg/mL) as described in Material and methods. Following cortactin immunoprecipitation, immunoblotting with anti-phosphotyrosine antibodies (clone 4 G10) or anti-cortactin antibodies detected tyrosine-phosphorylated cortactin or total amount of cortactin, respectively. Lanes a, d: basal condition; lanes b, e: ICAM-1 cross-linking; lanes c, f: sequential ICAM-1 and PECAM-1 cross-linking. Panels (b–e) IFN-γ-treated RBE4 cells were treated or not with calpeptin (150 μg/mL for 30 min) prior to ICAM-1 cross-linking. Cells were fixed and actin was stained by using 0.1 μg/mL of tetramethylrhodamine isothiocyanate-labeled phalloidin. Panel (b) Basal condition. Panel (c) ICAM-1 cross-linking. Panel (d) Basal condition after calpeptin treatment. Panel (e) ICAM-1 cross-linking after calpeptin treatment.

Article Snippet: Generation of HBMEC stable transfectants: HBMECs were transfected with either the pcDNA3.1-SHP-2 (human wild type) or pcDNA3.1-DnSHP-2 (Dominant negative) or “empty” plasmid (“mock”) kindly provided by Dr C. Nahmias ( Rivard et al. 1995 ) using the nucleofector system developed by Amaxa Inc (Gaithersburg, MD, USA).

Techniques: Incubation, Immunoprecipitation, Western Blot, Staining, Labeling

Journal: Journal of Neurochemistry

Article Title: PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells 2

doi: 10.1111/j.1471-4159.2007.04782.x

Figure Lengend Snippet: Cross-talk between ICAM-1 and PECAM-1-coupled signaling pathways. SHP-2 is positively involved in ICAM-1 signaling, upstream of the tyrosine phosphorylation of cortactin by Src and of RhoA-mediated actin cytoskeleton rearrangements. In this study, we show that PECAM-1 engagement, which induces a strong interaction with SHP-2, drastically inhibits these two distinct ICAM-1 activated pathways. Together, we propose that SHP-2 might be a key player at the crossroads between ICAM-1 and PECAM-1 signalings in endothelial cells.

Article Snippet: Generation of HBMEC stable transfectants: HBMECs were transfected with either the pcDNA3.1-SHP-2 (human wild type) or pcDNA3.1-DnSHP-2 (Dominant negative) or “empty” plasmid (“mock”) kindly provided by Dr C. Nahmias ( Rivard et al. 1995 ) using the nucleofector system developed by Amaxa Inc (Gaithersburg, MD, USA).

Techniques: