Fig 1: Calpain, through activation of PTP1B, regulates vinculin conformational change. Scanning angle interference microscopy for vinculin Z-position in MDCK cells treated as follows: (A,B) no treatment, with PTP1B inhibitor (RK682 10 µg/ml), with calpain-1 (CAPN1) inhibitor (ALLN, 10 and 50 µM), and with calpain-2 (CAPN2) inhibitor (ALLM, 10 and 50 µM); (C,D) in MDCK cells transfected with dominant active SrcY529F mutant and in MDCK vinculin KD cells with vinculin phosphomimetic mutant Y1065E and non-phosphorylatable mutant Y1065F, not treated and after drugs treatment (vanadate 10 µM and nocodazole 10 µM, 1 h). (E,F) Z-position of vinculin phosphomimetic mutant Y822E and non-phosphorylatable mutant Y822F. (A,C,E) Notched box plots for the Z-position of vinculin in E-cadherin-based adhesions. Notched box plots indicate first and third quartiles, median, and confidence intervals; whiskers, 5th and 95th percentiles. Median center values are indicated below each box plot (red). n values are shown above each box plot and indicate the numbers of adhesions (number of ROIs, black). Numbers of cells are indicated in blue. Red dotted lines indicate 5th and 95th percentiles of vinculin wt C-terminus for reference. (B,D,F) Topographic maps of protein Z-positions (nanometers) of the indicated proteins in E-cadherin-based adhesions of MDCK cells. Images were acquired by TIRF microscope with a 60x objective, 1.49NA Plan-Apo (Nikon). Scale bar, 5 µm. (G) Diagram showing the dual mechanism by which tension can modulate vinculin conformational switch.
Fig 2: PTP1B colocalizes with ß-catenin at E-cadherin-mediated adhesions and is associated with the ER. MDCK cells transfected with calnexin-mEmerald seeded on E-cadherin biomimetic substrate in control (A) and after 1 h of nocodazole (10 µm) treatment (C). Top panel of (A,C), representative images of MDCK cells transfected with calnexin-mEmerald (for ER identification, in cyan) and stained for ß-catenin (red), and PTP1B (green). In the zoom image (white box in the ß-catenin image), the colocalization of ß-catenin (red) and PTP1B (green) is visible. In the enhanced contrast image (white dotted box in the PTP1B image), the colocalization of PTP1B (purple) and ER (cyan) is visible. Bottom panel of (A,C), merged images for ß-catenin/ER, ß-catenin/PTP1B, and ER/PTP1B. In (B,D), line profiles of the dotted lines in PTP1B/ß-catenin merged images, for ß-catenin and PTP1B, respectively. Images were acquired by spinning disk confocal microscope with a ×60 objective, 1.49NA Plan-Apo (Nikon). Scale bar, 10 µm.
Fig 3: Integration of biochemical signals by the vinculin molecular clutch is regulated by PTP1B dephosphorylation, modulated by calpain cleavage. MDCK cells were cultured on fibronectin-coated substrate to form confluent monolayers, with cell–cell contacts labeled by ZO1-mEmerald. Cells were treated with pharmacological inhibitors as indicated. (A) Montage of consecutive frames (interval: 2 s) shown with junction excision at t = 0 s (asterisk). Recoiling edges of the junctions (red circles) were used to quantify the recoil trajectory in (B,C). Untreated MDCK (ctrl) were compared with MDCK cells treated with calpain 1 (CAPN1) inhibitor (ALLN, 50 µM for 1 h), calpain 2 (CAPN2) inhibitor (ALLM, 50 µM for 1 h), and PTP1B inhibitor (RK682, 10 µg/ml for 1 h). Scale bar, 5 µm. Junction recoil (B) and initial recoil rate (C) upon laser ablation of native cell–cell junctions in MDCK epithelial monolayer (wt) and with the indicated pharmacological treatments. Colors of plots in (C) correspond to bar graphs in (B). Ablation occurred at 0 s. Images were acquired by Nikon A1R MP laser scanning confocal microscope with 60x obj 1.49 NA. Error bars: S.E.M. n values: 15 (ctrl); 17 (+ RK682); 17 (+ ALLN); 11 (+ ALLM). ***P < 0.0005.
Fig 4: The crystal structure of PTP1B in complex with bisphosphorylated JAK peptides indicates a preference for the second phosphotyrosine.a Structure of PTP1B C215A/D181A/Q262A mutant bound to JAK2 and TYK2 activation loop peptides compared to IRK. Arg 47 of PTP1B interacts with the first phosphotyrosine of both JAK2 and TYK2 (phosphotyrosine 1007 of JAK2 and phosphotyrosine 1045 of TYK2), while the second phosphotyrosine (phosphotyrosine 1008 of JAK2 and phosphotyrosine 1046 of TYK2) sits in the catalytic pocket. b Detailed view of the interactions between the JAK peptides and PTP1B with 2Fo-Fc composite omit map with simulated annealing, contoured to 1 s. Maps are shown only for the JAK2 and TYK2 activation loop peptide residues.
Fig 5: PTP1B binds both monophosphorylated forms of the activation loop.Structures of PTP1B D1818A/Q262A/C215A in complex with pYpY, pYY and YpY JAK2 activation loop peptides. For both monophosphorylated peptides, the phosphorylated tyrosine occupies the catalytic pocket.
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