The TC-PTP phosphatase does not dephosphorylate PTP1B substrates. T-REx-293 cells were incubated for 24 h with doxycycline to induce the expression of PTP1B (A and B) and TC-PTP (A and C) phosphatases. A, different amounts of total extract expressing both PTP1B and TC-PTP were loaded onto the gel and the blot was revealed with anti-FLAG antibody. B and C, cells were stimulated with EGF for 5 min before cell lysis. 1 mg of the protein extract of each sample was immunoprecipitated with 4G10 antibody. The input material (Input) and the phosphorylated proteins (IP anti-4G10) were immunoblotted with antibodies against the indicated proteins. The phosphorylation levels of the proteins were normalized with the phospho-Grb2 signal and plotted as a bar diagram.

PLC-γ1, Gab1, and SHP2 are dephosphorylated by PTP1B upon insulin stimulation. HEK293 cells were transiently transfected with FLAG-tagged PTP1B (lanes 4–6) and FLAG vector as negative control (lanes 1–3). After serum starvation for 18 h, cells were stimulated with 100 ng/ml of insulin for 5 (lanes 2 and 5) and 20 min (lanes 3 and 6). As control we used unstimulated cells (lanes 1 and 4). A, total extracts were revealed with anti-4G10 antibody to monitor tyrosine phosphorylation after insulin induction (WB: anti-4G10) and with PTP1B specific antibody (WB: anti-PTP1B) to confirm phosphatase overexpression in lanes 4–6. WB, Western blot. B, the lysates were also revealed with antibodies against PLC-γ1, Gab1, SHP2, Grb2, and tubulin to detect protein levels in the cell extract. C, cell lysates were immunoprecipitated with anti-PLC-γ1, Gab1, and SHP2 antibodies. The blots were incubated with protein-specific (upper panels) or with anti-phosphotyrosine antibodies (WB: 4G10) to monitor the phosphorylation levels of the immunoprecipitated proteins (lower panels). After normalization of the immunoprecipitated proteins, phosphorylation levels were quantified and plotted. D, cell extracts were immunoprecipitated with anti-4G10 and revealed with anti-PLC-γ1, anti-Gab1, anti-SHP2, and anti-Grb2 antibodies.

Model of the proposed mechanism by which PTP1B controls ERK activity. The schematic only shows the proteins and the interactions that are relevant for the novel mechanism described in this article. In principle, PTB1B could indirectly associate with Gab1 via interactions mediated by the two SH3 domains of Grb2. However, we have chosen to show a complex that links PTP1B to Gab1 via two intermediate interactions mediated by the SH2 domains of Grb2 and SHP2. Although we have no direct proof of the existence of such a tetracomplex, such an interaction topology, based on established binary interactions can better explain our observation that the association between PTP1B and Gab1 is EGF dependent. The SHP2 phosphatase is shown to activate ERK by dephosphorylating Gab1 phosphotyrosine 317 thus inhibiting formation of a Gab1-RasGAP complex (45). Here we add that this positive regulation of the ERK pathway can be shut down by the PTP1B-mediated dephosphorylation of the SHP2 docking sites on Gab1 (Tyr-627 and Tyr-659). PR stands for proline rich, PH for pleckstrin homology domain, SH for Src homology domain, TK for tyrosine kinase, and PTP for phosphotyrosine phosphatase domain.

Schematic illustration of the strategy used to discover potential PTP1B substrates. A, high density peptide chip technology is used to identify the phosphopeptides that have the capacity of binding the trapping mutant catalytic domain of PTP1B. The results are used to create a PSSM to rank the human tyrosine-phosphorylated proteins according to the likelihood of being PTP1B substrates in vitro. B, a second feature used in the prediction strategy is WID. We first compiled a weighted human interactome by combining the interactions imported from four major protein-protein interaction databases: DIP, HPRD, IntAct, and MINT. For each protein in the human interactome we next calculated WID, a weighted graph distance from PTP1B. C, because the two features are largely independent we integrated them by a naive Bayesian approach. The Bayesian framework ranked each phosphorylated human protein with a score reflecting the probability of being a physiological target of PTP1B. D, finally we used a Pathway filter to retain only the substrates that had already been described as playing a role in the EGF and insulin pathways.

Knock down of PTP1B expression by siRNA results in an increase in the level of tyrosine phosphorylation of PLC-γ1, Gab1, and SHP2. HEK293 stably expressing PTP1B siRNA (lanes 3 and 4) or a scrambled control (lanes 1 and 2) were transiently transfected with a plasmid encoding the constitutively active Src Y527F (lanes 2 and 4) or with the empty vector (lanes 1 and 3). A, protein extracts were immunoblotted with the 4G10 antibody, to monitor the phosphotyrosine profile of HEK293 cells, with an antibody against the Src kinase to verify ectopic expression of the constitutively active mutant (lanes 2 and 4), with an anti-PTP1B antibody, to monitor the endogenous PTP1B expression levels and antitubulin, as a loading control. B, 1 mg of protein extract was immunoprecipitated with the anti-phosphotyrosine antibody 4G10 (IP: anti-4G10). The input cell extracts and the IP material were immunoblotted (WB) with antibodies against PLC-γ1, Gab1, SHP2, and Grb2. The amount of IgG was used as loading control. The phosphorylation levels of the indicated proteins were normalized and plotted as a bar diagram.

PTP1B directly dephosphorylates Gab1 and PLC-γ1 but not GRB2 in vitro. A, the PTP1B catalytic domain was expressed in bacteria, purified, and incubated with p-nitrophenyl phosphate substrate in assay buffer to demonstrate phosphatase activity. The increase in the absorbance at 405 was plotted as a function of time, and the specific activity was calculated by applying the Lambert-Beer law. B, D, and F, T-REx-293 cells were transiently transfected with 10 μg of a plasmid directing the synthesis of the Src mutant Y527F, a constitutively active mutant of SRC kinase. After cell lysis the protein extracts were immunoprecipitated with anti-Gab1, anti-PLCγ-1, or anti-Grb2 antibodies. The immunopurified proteins were incubated with 0.3 μg of GST-PTP1B catalytic domain or GST alone, for the indicated times, at 30 °C, in reaction buffer. The tyrosine phosphorylation levels and the immunoprecipitated proteins were revealed, respectively, with anti-4G10 and anti-Gab1, PLCγ-1, and Grb2 antibodies (C, E, and G). The band intensities were quantitated and the ratios of phosphoproteins (p-Gab1, p-PLCγ-1, and p-Grb2) versus total proteins (Gab1, PLCγ-1, and Grb2) are represented as bar graphs.

PTP1B negatively affects SHP2 recruitment by phosphorylated Gab1 and ERK phosphorylation. A, HEK293 cells were transiently transfected with FLAG-tagged PTP1B (lanes 2 and 4) or with the corresponding amount of empty FLAG vector (lanes 1 and 3) and stimulated with EGF for 5 min (lanes 3 and 4). 1 mg of protein extract was immunoprecipitated with anti-Gab1 antibody. The IP material was immunoblotted with antibodies against Gab1 (to control Gab1 immunoprecipitation), 4G10 (to monitor Gab1 phosphorylation level), SHP2 (to verify its interaction with Gab1), and the purified SH2 domains of SHP2 (to verify the interaction with the phosphotyrosines of Gab1). The amount of immunoprecipitated Gab1 was used as a reference to quantify the fraction of Gab1 phosphorylated in the four different experimental conditions. In the bar diagram in A, we have quantified the amount of phospho-Gab1 (to verify EGF dependent phosphorylation), the amounts of SHP2 and SH2 domains bound to Gab1 (to quantify the amount of SHP2 bound to phospho-Gab1 in lane 3 and the decrease of interaction due to Gab1 dephosphorylation by PTP1B in lane 4). The input was revealed with antibodies against Gab1, SHP2, PTP1B, and tubulin. The anti-β-tubulin antibody was used as a loading control and the anti-PTP1B antibody was used to verify PTP1B transfection. The protein extracts were revealed also with anti-phospho-p42–44 MAPK-ERK1/2, to control the EGF-induced activation of ERK1/2 (lane 3) and the decreased ERK phosphorylation subsequent to PTP1B expression (lane 4). The ratio of phosphorylated p42–44 MAPK to total ERK1/2 protein is plotted. B, T-REx-293 cells were incubated for 24 h with doxycycline to induce the expression of PTP1B phosphatase (lanes 3). Cells were induced 5 min with EGF (lanes 2 and 3). 1 mg of the protein extracts was immunoprecipitated with Gab1 antibody. The input and the Gab1-bound proteins (IP material) were immunoblotted with antibodies against Gab1, SHP2, and 4G10 (left). The expression of PTP1B was revealed with anti-FLAG antibody (lane 3). Bar diagram in B, the amount of immunoprecipitated Gab1 was used as a reference to quantify the SHP2 fraction bound to phosphorylated Gab1 (lane 2) to compare with the SHP2 bound to Gab1 in PTP1B overexpression conditions (lane 3). C, to check the co-immunoprecipitation of phospho-Gab1 when SHP2 is immunoprecipitated in the presence of low level of PTP1B, T-REx-293 cells were incubated for 24 h with doxycycline to induce expression of PTP1B (lanes 1 and 2). Five minutes before cell lysis, cells were stimulated with EGF (lanes 2 and 3). 1 mg of the protein extract of each sample was immunoprecipitated with anti-FLAG antibody (for PTP1B) and 1 mg was used to immunoprecipitate Gab1 protein. The input material (Input) as well as the IP with anti-FLAG (PTP1B) antibody were immunoblotted with antibodies against FLAG, Gab1, and Grb2. The IP with anti-Gab1 antibody were revealed with 4G10 antibody to detect the phosphorylation level of Gab1. D, inhibition of PTP1B activity increases ERK phosphorylation and improves co-immunoprecipitation of Gab1 with SHP2, although not affecting its co-immunoprecipitation with Grb2. T-REx-293 cells were incubated for 24 h with doxycycline to induce the expression of PTP1B (lanes 3 and 4) and serum starved for 20 h. An aliquot of 293 cells were treated with 250 μm of the PTP1B inhibitor (Calbiochem compound N0539741) for 1 h (lane 4) and induced with EGF for 5 min (lanes 2–4). Protein extracts were revealed with antibodies against the indicated proteins. The protein extracts were also immunoblotted with anti-phospho-p42–44 MAPK-ERK1/2, to monitor the activation of ERK1/2. 1 mg of protein extract was immunoprecipitated with an anti-Gab1 antibody and revealed with 4G10 antibodies (to monitor Gab1 phosphorylation levels), anti-Gab1 (to control Gab1 immunoprecipitation), and anti-SHP2, to verify its interaction with Gab1. The fractions of phosphorylated p42–44 MAPK and phosphorylated Gab1 are plotted as bar diagrams together with the fraction of SHP2 bound to Gab1.

PTP1B substrate prediction. A, the PTP1B substrate specificity was investigated by probing a high-density phosphopeptide chip with the substrate-trapping mutant of the PTP1B catalytic domain. Corresponding subarray regions (∼2 × 2 mm) are enlarged to demonstrate the reproducibility of the signal. B, two Sample Logo representation of PTP1B specificity. The logo is divided into two parts. At each position of the top diagram the residues (one letter code) that are enriched in the “positive peptide set” are represented with a font size relating to their enrichment factor. Conversely, the bottom diagram contains the residues that are enriched in the peptide sequences of the negative set. C, the performance of the PSSM was evaluated by a 10-fold cross-validation and by plotting a ROC curve, using as positive dataset the 300 binding and as negative dataset the 247 non-binding peptides identified by probing the phosphopeptide chip with the trapping mutant domain of PTP1B. D, correlation between PTP1B phosphatase activity and PSSM values. The phosphatase activity of PTP1B when assayed with 37 different peptides (21) was plotted against the peptide PSSM score. E, PLC-γ1, Gab1, and SHP2 bind the substrate-trapping mutant of PTP1B. Bacterially expressed GST, PTP1B trapping mutant domain (GST-PTP1B-DA), or wild type PTP1B catalytic domain (GST-PTP1B-WT), were used in pulldown assays by mixing with 1 mg of lysate from EGF-induced HEK293 cells. The affinity-purified proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with anti-phosphotyrosine antibody 4G10 (left) or protein-specific antibodies (right). An empty lane in both gel separates the input (lane 1) from lane 2, containing the GST pull down (negative control). Lane 3 displays the proteins bound to PTP1B substrate-trapping (the putative PTP1B substrates) and lane 4 displays the proteins that remain bound to wild type PTP1B. Lane 5, in both gels, show proteins immunoprecipitated with the anti-phosphotyrosine antibody 4G10 (to check phosphorylation level). F, quantitation of the pull down in panel E. The bar graph on the left represents the fraction of each protein that is phosphorylated (i.e. immunoprecipitated by the anti-phosphotyrosine antibody). The graph on the right represents the fraction of phosphorylated protein that is pulled down by the PTP1B substrate trapping mutant. G, the curves in the figure compare the ROCs of three differently constructed predictive models: the blue and green curves refer to models built on a single feature (PSSM or WID, respectively), whereas the red curve represents the performance of Bayesian models obtained by integrating both the PSSM and WID scores. The curves shown have been obtained by averaging 10 independently trained classifiers of the same kind by a bootstrapping approach. The AUC distributions of the 10 different models of the three predictors had a standard deviations of 0.02 or less.