Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: SHP-2 is activated in response to force on E-cadherin. (A–G) MCF10A cells, or MCF10A cells expressing shRNAs against SHP-2 (shSHP-2) or a scramble shRNA sequence (scSHP-2) were incubated with paramagnetic beads coated with IgG or E-cadherin extracellular domains (Ecad). Cells were left resting (no force, NF) or tensile force was applied (+), and the cells were lysed immediately (1 min) or at the indicated times (Min. after Force). (A–C) SHP-2 is activated by force, and loss of SHP-2, E-cadherin or mechanical signaling prevents activation. Lysates were immunoblotted for SHP-2 Y542 phosphorylation (pSHP-2) or total SHP-2. In C, the cells were pre-incubated with a myosin II inhibitor [blebbistatin (Blebbi)] or E-cadherin function-blocking antibody (HECD-1) prior to application of force. (D,E) Vinculin is phosphorylated when SHP-2 is inactive and dephosphorylated when SHP-2 is active. Lysates from the cells were immunoblotted with phospho-specific antibodies that recognize Y822 vinculin (pY822) or total vinculin. (F,G) SHP-2 is recruited to the cadherin adhesion complex in response to force. The magnetic beads were recovered, and co-precipitating levels of pSHP-2 were examined. In G, the cells were pre-treated with a myosin II inhibitor (Blebbi) or an E-cadherin function-blocking antibody (HECD-1) prior to application of force. (H,I) Shear stress elicits similar responses in SHP-2 activation and vinculin Y822 phosphorylation as tensile force. Shear stress was applied to cells, the cells were lysed at the indicated times, and the lysates were immunoblotted for pSHP-2 or total SHP-2 (H), or pY822 or total vinculin (I). The graphs beneath the immunoblots in all panels represent the quantification of a minimum of three independent experiments±s.e.m. *P<0.05.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Expressing, shRNA, Sequencing, Incubation, Activation Assay, Blocking Assay, Magnetic Beads, Shear, Western Blot

Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: SHP-2 specifically targets phospho-Y822 vinculin. (A) Vinculin binds a SHP-2 trapping mutant. MCF10A cells were left resting (−) or pre-treated with pervanadate and then lysed. The lysates were incubated with a substrate-trapping mutant, GST-SHP-2 (D425A/C459A). The co-precipitating levels of vinculin were monitored by immunoblotting, and the levels of the trapping mutant were visualized by Coomassie Blue staining of the gel. (B,C) Vinculin binding to the SHP-2 trapping mutant is blocked by mutation of Y822F. GFP fusions of wild-type (WT), Y100F, Y822F or Y1065F vinculin were expressed in MCF10A cells, and the levels of each expressed protein were examined by immunoblotting with tubulin as a loading control in B. In C, lysates from the indicated cell lines were incubated with the SHP-2 trapping mutant and the products of the reactions were examined as described in A. (D) Force increases vinculin trapping by SHP-2. Cells were left resting (no force, NF) or tensile force was applied and the cells were lysed immediately (1 min) or 10 min later. Substrate trapping was measured as described in A. (E) SHP-2 dephosphorylates Y822 vinculin in vitro. Cells were left untreated or treated with pervanadate. Vinculin was immunoprecipitated, and the immunoprecipitates were subjected to an in vitro phosphatase assay in the presence (+) or absence (−) of recombinant SHP-2. Levels of vinculin phosphorylated at Y822 (pY822) relative to total immunoprecipitated vinculin were monitored by immunoblotting. Graphs adjacent to each image represent the quantification of a minimum of three independent experiments±s.e.m. *P<0.05.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Mutagenesis, Incubation, Western Blot, Staining, Binding Assay, In Vitro, Immunoprecipitation, Phosphatase Assay, Recombinant

Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: Inhibition of SHP-2 cells elevates mechanotransduction. (A) A schematic of the E-cadherin-mediated cell-stiffening cascade. Force on E-cadherin activates a mechanosignaling pathway, whereby Abl phosphorylates vinculin at Y822. Vinculin Y822 phosphorylation is essential for E-cadherin-dependent RhoA-mediated contractility, which culminates in phosphorylation of MLC and heightened actomyosin contractility. (B) SHP-2 expression is inhibited in cells expressing shRNAs. Two mass populations of cells expressing shRNAs against SHP-2 (shSHP-2 and shSHP-2.2) were generated and examined by immunoblotting for SHP-2 levels relative to a loading control (p34-Arc subunit of the Arp2/3 complex). The graphs below each image represent the quantification of a minimum of three independent experiments±s.e.m. (C,D) Mechanotransduction is elevated in cells with depressed SHP-2 levels. Lysates from parental or shSHP-2 cells were immunoblotted for vinculin phosphorylated at Y822 (pY822) or total vinculin in C, and MLC phosphorylated at T18 and S19 (pMLC) or total MLC in D. The graphs below each image represent the quantification of a minimum of three independent experiments±s.e.m. (E–G) SHP-2 inhibition increases actin reinforcement. MCF10A parental or shSHP-2 cells were left resting or subjected to shear stress as described in the Materials and Methods. The cells were fixed and stained with antibodies against β-catenin, or with Texas-Red Phalloidin, and examined by confocal microscopy (E). Graphs beside images represent the average corrected fluorescence intensity of F-actin (G) or β-catenin (F) in the cell–cell junctions. Fifty junctions were quantified for each condition. Box-and-whisker plots represent the 10th, 25th, 50th, 75th and 90th percentiles. *P<0.05. Scale bar: 10 µm.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Inhibition, Expressing, Generated, Western Blot, Shear, Staining, Confocal Microscopy, Fluorescence, Whisker Assay

Journal: Journal of Cell Science

Article Title: SHP-2 is activated in response to force on E-cadherin and dephosphorylates vinculin Y822

doi: 10.1242/jcs.216648

Figure Lengend Snippet: Mutation of the residues N-terminal of Y822 creates a vinculin mutant that is constitutively phosphorylated. (A) A schematic depicting the amino acid sequences surrounding vinculin Y822. SHP-2 prefers substrates with acidic residues at the −3 and −2 positions. In vinculin, the −2 and −3 positions are occupied by the acidic residues, S and D. (B) The mutant vinculins were expressed at similar levels in MCF10A cells. Full-length vinculin harboring the indicated mutations was stably expressed as a fusion with GFP in MCF10A cells. The levels of expression were examined by immunoblotting for GFP and tubulin as a loading control. (C) The mutant vinculins do not bind SHP-2. The cells expressing the mutant vinculins were pre-treated with pervanadate for 30 min. Lysates were incubated with GST-SHP-2 (D425A/C459A) and the co-precipitating levels of GFP-vinculin were monitored by immunoblotting. (D) Mutant vinculins retain phosphorylation at Y822. The mutant vinculins were immunoprecipitated. Levels of vinculin Y822 phosphorylation were monitored by immunoblotting with antibodies against phospho-Y822, and then the blots were stripped and re-probed for total vinculin levels. (E) Mutant vinculins are recruited to the E-cadherin adhesion complex. The mutant vinculins were immunoprecipitated. Levels of co-precipitating E-cadherin were monitored by immunoblotting. The graphs in each panel represent the quantification of at least three independent experiments±s.e.m. *P<0.05.

Article Snippet: Immunoprecipitates were washed three times in 1× phosphatase buffer, resuspended in 2× phosphatase buffer (50 mM HEPES, pH 7.4, 0.2 mM EDTA, 10 mM DTT, 200 μg/ml BSA, pH 7.45), and incubated with 1.0 µg recombinant SHP-2 (R&D Systems, 1894-SH) at 30°C for 30 min.

Techniques: Mutagenesis, Stable Transfection, Expressing, Western Blot, Incubation, Immunoprecipitation

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1/2 are required for insulin-activated IR endocytosis. a HepG2 cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP antibodies. b Quantification of the ratios of PM and IC IR-GFP signals of cells in ( a ) (mean ± SD; *p<0.0001). c Domains and YXXΦ motifs of human IRS1 and mouse IRS2. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. AP2M1- and SHP2-binding regions are indicated. YXXΦ motifs and phosphotyrosine sites of IR for SHP2 binding are shown as blue and red bars, respectively. The MAPK phosphorylation sites are labeled as green letters in the sequences. d 293FT cells stably expressing IR-GFP WT were transfected with the indicated siRNAs or siRNA-resistant Myc-IRS1, serum starved, treated without or with 100 nM insulin for 5 min, and stained with anti-GFP (IR; green), anti-Myc (IRS1; red), and DAPI (blue). (3YA, Y612A/Y632A/Y662A; 3YF, Y612F/Y632F/Y662F; 3SA, S616A/S636A/S666A; 3SD, S616D/S636D/S666D; Y2A, Y1179A/Y1229A). e Quantification of the ratios of PM and IC IR-GFP signals of cells in ( d ) (mean ± SD; *p<0.0001). f 293FT cells were serum starved and treated without or with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP, and IgG IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. g Serum-starved primary hepatocytes were treated with DMSO or 10 µM SHP099 for 2 h and treated with 100 nM insulin for 5 min. Total cell lysate (TCL), anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: IRS1 promotes IR endocytosis and interacts with AP2. a Western blot analysis of cell lysates in . Asterisks indicate non-specific bands. b Domains and YXXΦ motifs of human IRS1. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. IRS1 fragments that can or cannot bind to AP2M1 are presented as red or black lines, respectively. YXXΦ motifs and phosphotyrosine sites for SHP2 binding are presented as blue and red bars, respectively. c Binding of IRS1 WT and mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with anti-Myc (IRS1) antibodies and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=3 independent experiments). d Binding of IRS1 WT and truncation mutants to GST or GST-AP2M1. Input and protein bound to beads were blotted with the indicated antibodies. The relative band intensities are shown below (n=2 independent experiments). e Sequence alignment of a conserved region in IRS1/2. Three YXXΦ motifs are boxed with red dashed lines. The phosphorylation sites of IR and MAPK are indicated as red and blue dots, respectively.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Western Blot, Binding Assay, Staining, Sequencing

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: The SHP2-MAPK pathway promotes insulin-activated IR endocytosis. a HepG2 cells expressing IR-GFP WT were starved, treated with the indicated inhibitors for 2 h, treated without or with 100 nM insulin for 20 min, and stained with anti-GFP (IR; green) and DAPI (blue). b Quantification of the ratios of PM and IC IR-GFP signals of cells in (A) (mean ± SD; *p<0.0001). c Binding of IRS1 peptides to AP2M1 (residues 160-435). Input and proteins bound to IRS1-peptide beads were analyzed by SDS-PAGE and stained with Coomassie (CBB). The relative band intensities are shown below (mean ± SD; n=4 independent experiments). d Isothermal titration calorimetry (ITC) analysis of binding between IRS1 peptides and AP2M1 (residue 160-435), with K d indicated. e The IRS1 peptides were incubated with active SHP2 for the indicated durations, spotted onto membranes, and detected with the anti-pY612-IRS1 antibody. f Quantification of the relative SHP2 activity in ( e ) (mean ± SD; n=4 independent experiments; *p<0.0001). g Model of the regulation of insulin-activated IR endocytosis by a phosphorylation switch on IRS1/2. Insulin-bound IR phosphorylates itself and IRS1/2, and activates the PI3K-AKT and MAPK pathways. SHP2 acts upstream of RAS-RAF and promotes the activation of MAPK pathway. p31 comet binds to the IR-bound MAD2 and blocks IR-AP2 association to prevent premature IR endocytosis. In feedback regulation, activated ERK1/2 phosphorylate S616 and other sites on IRS1. SHP2 binds to the C-terminal phospho-tyrosine site on IRS1 and dephosphorylates pY612 of the doubly phosphorylated IRS1 (pY612/pS616), thus promoting IRS1-AP2M1 association. p31 comet is released from MAD2 by an unknown mechanism, allowing the assembly of an MCC-like complex on IR. MAD2- and IRS1/2-dependent AP2 recruitment and clustering trigger clathrin-mediated IR endocytosis. h Ribbon diagram of the crystal structure of AP2M1 (residues 160-435) bound to pS-IRS1. pS-IRS1 is shown as sticks. i Surface drawing of AP2M1, with pS-IRS1 shown as sticks. j A close-up view of the surface drawing of AP2M1 colored by its electrostatic potential (blue, positive; red, negative; white, neutral). pS-IRS1 is shown as sticks.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Expressing, Staining, Binding Assay, SDS Page, Isothermal Titration Calorimetry, Incubation, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: SHP2 inhibition delays IR endocytosis and improves insulin sensitivity in mice. a , b Glucose tolerance test ( a ) and insulin tolerance test ( b ) of male mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. At 1 day after the last drug administration, experiments were performed. Vehicle, n=12; SHP099, n=10; mean ± SEM. c Body weight of mice administered vehicle or SHP099 at 7 days post administration. Mean ± SD. d HFD-fed mice were administered vehicle or SHP099 for 5 days. The mice were fasted overnight and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. e Quantification of the ratios of plasma membrane (PM) and intracellular compartments (IC) IR signals of the livers in ( d ) (mean ± SD; *p<0.0001). f - h The levels of fasting serum insulin ( f ) and C-peptide ( g ), and the ratio of C-peptide:insulin ( h ) in mice fed normal chow or HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. i HepG2 cells stably expressing IR-GFP were transfected with CEACAM1 siRNAs, serum starved, treated without or with 100 nM insulin form 5 min, and stained with anti-GFP and DAPI. Quantification of the ratios of PM and IC IR-GFP signals of cells was shown (mean ± SD). j Western blot analysis of cell lysates in ( i ). k Model of the regulation of insulin-activated IR endocytosis by CEACAM1, the MAD2–CDC20– BUBR1 module, and the SHP2-IRS1/2 module.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Staining, Stable Transfection, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Depletion of SHP2 by shRNA delays IR endocytosis and improves insulin sensitivity in mice. a The level of SHP2 in liver, skeletal muscle and epididymal WAT from mice fed HFD for 5 weeks. The mice were injected with AAV-control (Ctrl) or SHP2 shRNA. At 17 days after injection, the mice were fasted overnight and injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. WAT and skeletal muscle were collected at 2 min and 3 min after the indicated time points, respectively. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b , c Glucose tolerance test ( b ) and insulin tolerance test ( c ) in mice injected with AAV-Ctrl or AAV-SHP2 shRNA and fed HFD. Experiments were performed at 2 weeks after injection. n=6; mean ± SEM. d Body weight in HFD-fed mice injected with AAV-Ctrl or AAV-SHP2 shRNA. Mean ± SD. e HFD-fed mice were injected with AAV-Ctrl or AAV-SHP2. At 17 days after injection, the mice were fasted overnight and injected with or without 1U insulin via inferior vena cava. The livers were collected at the indicated time points and the sections were stained with anti-IR (red) and DAPI (blue). Scale bars, 5 µm. f Quantification of the ratios of PM and IC IR signals of the livers in ( e ) (mean ± SD; *p<0.0001).

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: shRNA, Injection, Western Blot, Staining

Journal: bioRxiv

Article Title: Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

doi: 10.1101/419911

Figure Lengend Snippet: Mitotic regulators and SHP2 promote feedback inhibition of IR. a Insulin signaling in the liver from mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 5 days, fasted overnight, and administered vehicle or SHP099 once more. At 2 h after the last administration, the mice were injected with or without 1 U insulin via inferior vena cava. The livers were collected at the indicated time points. Lysates were prepared from these tissues and subjected to quantitative immunoblotting with the indicated antibodies. b Quantification of the blots in ( a ). Mean ± SD; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. c Targeting feedback regulation of IR endocytosis for diabetes treatment. Left panel depicts the feedback regulation of IR endocytosis by ERK1/2 and SHP2 during unperturbed insulin signaling. Right panel illustrates the mechanism by which SHP2 inhibitor (SHP099) or shRNA blocks growth-promoting IR signaling and IR endocytosis, and prolongs insulin signaling through the PI3K-AKT pathway, which controls metabolism.

Article Snippet: Active SHP2 (2.9 µM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, 65 ng/µl BSA] was incubated with IRS1 peptides (2.6 mM) at 37°C for the indicated time points.

Techniques: Inhibition, Injection, Western Blot, shRNA