vitro phosphatase assays active shp2  (SignalChem)

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

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 (siLUC, n = 26; siLUC + Ins, n = 42; si-IRS1, n = 41; si-IRS1, n = 26; si-IRS2, n = 20; si-IRS2 + Ins, n = 10; si-IRS1/2, n = 48, si-IRS1/2 + Ins, n = 66; si-IRS1/2 + IRS1, n = 14; si-IRS1/2 + IRS1 + Ins, n = 14; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). 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, the IRS1 Y612A/Y632A/Y662A triple mutant; 3YF, the Y612F/Y632F/Y662F triple mutant; 3SA, the S616A/S636A/S666A triple mutant; 3SD, the S616D/S636D/S666D triple mutant; 2YA, the Y1179A/Y1229A double mutant). Scale bar, 5 µm. e Quantification of the ratios of PM and IC IR-GFP signals of cells in d (siLUC, n = 49; siLUC + Ins, n = 87; si-IRS1/2, n = 46; si-IRS1/2 + Ins, n = 87; si-IRS1/2 + WT, n = 65; si-IRS1/2 + WT + Ins, n = 112; si-IRS1/2 + 3YA, n = 54; si-IRS1/2 + 3YA + Ins, n = 63, si-IRS1/2 + 3SA, n = 40; si-IRS1/2 + 3SA + Ins, n = 66; si-IRS1/2 + 3 SD, n = 42; si-IRS1/2 + 3 SD + Ins, n = 63; si-IRS1/2 + 3YF, n = 48; si-IRS1/2 + 3YF + Ins, n = 58; si-IRS1/2 + Y612A, n = 48; si-IRS1/2 + Y612A, n = 58; si-IRS1/2 + 1–864, n = 23; si-IRS1/2 + 1–864 + Ins, n = 35; si-IRS1/2 + 2YA, n = 28, si-IRS1/2 + 2YA + Ins, n = 42; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). 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. TCL, anti-IRS1 IP were blotted with anti-IRS1 and anti-AP2B1 antibodies. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Two Tailed Test, Binding Assay, Phospho-proteomics, Labeling, Mutagenesis

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

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). Scale bar, 5 µm. b Quantification of the ratios of PM and IC IR-GFP signals of cells in a (DMSO, n = 47; DMSO + Ins, n = 56; IRi, n = 50; IRi + Ins, n = 65; U0126, n = 96; U0126 + Ins, n = 111; PD0325901, n = 26; PD0325901 + Ins, n = 54; SHP099, n = 43; SHP099 + Ins, n = 49; GDC0941, n = 57; GC0941 + Ins, n = 48; AKTi, n = 65; AKTi + Ins, n = 96; BI2536, n = 26; BI2536 + Ins, n = 24; Reversine, n = 37; Reversine + Ins, n = 27; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). 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 ± s.d.; n = 5 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 ± s.d.; n = 4 independent experiments; **** p < 0.0001; two-tailed unpaired t test). 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 phosphotyrosine 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. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Expressing, Staining, Two Tailed Test, Binding Assay, SDS Page, Isothermal Titration Calorimetry, Residue, Incubation, Activity Assay, Phospho-proteomics, Activation Assay

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

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. For GTT ( a ), vehicle, n = 12; SHP099, n = 10; for ITT ( b ), vehicle, n = 11; SHP099, n = 10; mean ± s.e.m; two-tailed unpaired t test. c Body weight of mice administered vehicle or SHP099 at 7 days post administration. Mean ± s.d. d HFD-fed mice were administered vehicle or SHP099 for 5 days. The mice were fasted overnight and again administered vehicle or SHP099. 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 (vehicle, 0 min, n = 43; 1 min, n = 54; 5 min, n = 27; 15 min, n = 36 and SHP099, 0 min, n = 35; 1 min, n = 43; 5 min, n = 30; 10 min, n = 33; 15 min, n = 33; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). f – h The levels of fasting serum insulin ( f ) and C-peptide ( g ), and the ratio of C-peptide:insulin ( h ) in mice fed HFD for 5 weeks. The mice were administered vehicle or SHP099 for 6 days. Vehicle, n = 11; SHP099, n = 12. 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 (siLUC, n = 28; siLUC + Ins, n = 49; siCEACAM1 #1 + Ins, n = 27; siCEACAM1 #2 + Ins, n = 25; siCEACAM1 #3 + Ins, n = 35; siCEACAM1 #4 + Ins, n = 18; mean ± s.d.; **** p < 0.0001; two-tailed unpaired t test). j Western blot analysis of cell lysates in i . k Model of the regulation of insulin-activated IR endocytosis. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Inhibition, Two Tailed Test, Injection, Staining, Clinical Proteomics, Membrane, Stable Transfection, Expressing, Transfection, Western Blot

Journal: Nature Communications

Article Title: Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling

doi: 10.1038/s41467-019-09318-3

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 ± range; n = 2 independent experiments. 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. Source data are provided as a Source Data file

Article Snippet: Active SHP2 (2.9 μM, SignalChem) diluted in the phosphatase dilution buffer [50 mM imidazole, pH 7.2, 0.2% 2-mercaptoethanol, and 65 ng μl −1 BSA] was incubated with IRS1 peptides (2.6 mM) at 37 °C for the indicated time points.

Techniques: Inhibition, Injection, Western Blot, shRNA

Journal: Nature cell biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells.

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: Fig. 5 | ITGB1 is a substrate for PTP-PEST and Shp2. a,b, A malachite green assay for free phosphate release after incubation of phosphorylated/non- phosphorylated ITGB1 peptides with recombinant Shp2 (n = 5 independent replicates, each performed in triplicate) (a) or PTP-PEST (n = 4 independent replicates, each performed in triplicate) (b). The significance was assessed using a Kruskal–Wallis test with a Dunn’s correction for multiple comparisons. The data are presented as the mean ± s.e.m. c,d, Schematics of FRET experiments (left) using mRuby2-tagged ITGB1 and Clover-tagged PTPs. Representative FLIM–FRET images (right) and quantification of apparent FRET efficiency of MM231 cells with stable expression of either ITGB1(WT)–mRuby2 or ITGB1(YYFF)–mRuby2

Article Snippet: Each fragment (2,400 pmol per peptide per reaction) was incubated separately for 1 h at 37 °C with recombinant Shp2 (0.05 μg ml−1; R&D Systems, 1894-SH-100) or PTP-PEST (0.05 μg ml−1; SignalChem, P39-21G-10) in phosphatase buffer (HEPES buffer, pH 7.5 (50 mM)/EDTA (0.2 mM)/DTT (5 mM)/Triton X-100 (0.01%)), before incubation with Malachite Green Reagent (100 μl per reaction).

Techniques: Malachite Green Assay, Incubation, Recombinant, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The Toxoplasma secreted effector Tg WIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2

doi: 10.1007/s00018-024-05283-3

Figure Lengend Snippet: Phosphorylated Tg WIP binds to host cell tyrosine phosphatases Shp1 and Shp2. A Schematic showing the amino acid sequence of Tg WIP. Magenta color indicates the position of the three tyrosines in Tg WIP. Orange region represents the WRC interacting receptor sequence (WIRS) domain that could mediate Tg WIP’s interaction with the WAVE complex. Blue regions represent the proline rich regions (PRRs) that could mediate Tg WIP’s interactions with Nck and Grb2. B DCs were infected with parasites expressing wild-type Tg WIP ( Tg WIP WT ), or Tg WIP in which its SH2-binding motifs Y150 ( Tg WIP Y150A ), Y199 ( Tg WIP Y199A ), or both ( Tg WIP Y150A/Y199A ) are mutated to an alanine. Shown are the Western blots using phosphorylated tyrosine (pY) (1st membrane-probing antibody), HA (2nd membrane-probing antibody after stripping membrane), and Shp2 (3rd membrane-probing antibody after stripping membrane twice) antibodies on total lysate or immunoprecipitated Tg WIP. C The murine DC (DC2.4) and human foreskin fibroblast (HFF) cell lines were infected with type II ME49 Δtgwip complemented with HA-tagged wild-type Tg WIP ( Tg WIP WT ) for 4 h (intracellular) or until parasite egress (extracellular). Lysates were generated and Tg WIP immunoprecipitated. Shown are the Western blots using phosphorylated tyrosine (pY), HA, Shp1, and Shp2 antibodies, (probed in the same order as in C). D DC2.4 were treated with the Src kinase inhibitor Dasatinib at the indicated concentrations for 1 h and throughout infection with WT Toxoplasma . DMSO is the solvent control. Western blot analysis using antibodies against pY and HA. DCs were infected with Tg WIP WT or with either E Tg WIP Y150A or with F Tg WIP Y150A/Y199A Toxoplasma. Shown are the Western blots using antibodies against Shp2 in E and Shp1 in F . G-H In vitro phosphatase assay of Shp2 in G Shp1 in H incubated with recombinant Tg WIP, with or without prior phosphorylation by recombinant Src kinase. Reaction rates, represented by linear fluorescence changes over time, were normalized against the rate of Shp2 or Shp1 without Tg WIP. Data from 3 biological repeats are indicated by different colors, with each data point showing the average from three technical repeats. Bar graph represents the mean and standard error of the 3 biological repeats. ANOVA with Tukey’s post hoc test was used. n = 3; *** for p < 0.0005 against the first column). n.s.: not significant. I-J Coomassie blue-stained SDS-PAGE gel showing GST-tagged, Src-phosphorylated Tg WIP (GST-TgWIP P ) pulling down MBP-tagged full-length (FL) or individual SH2 domains of Shp2 in I or Shp1 in J . Loading controls contain 6 pmol of indicated prey proteins

Article Snippet: The impact of phosphorylated Tg WIP on both Shp1 and Shp2 activity was measured using a Shp2 activity assay kit (BPS bioscience, Cat# 79,330 [ ]), following the manufacturer’s instructions.

Techniques: Sequencing, Infection, Expressing, Binding Assay, Western Blot, Membrane, Stripping Membranes, Immunoprecipitation, Generated, Solvent, Control, In Vitro, Phosphatase Assay, Incubation, Recombinant, Fluorescence, Staining, SDS Page

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