Because of its involvement in modulating the outcome of numerous human disorders, PTP1B is the most intensively studied tyrosine phosphatase. Despite the wealth of available information, the network of interactions, and enzymatic substrates that have been assembled over the years is not sufficient to fully explain the physiological outcome of modulating the levels of this phosphatase in cells or living organisms. A variety of reports are consistent with the notion that the main role of PTP1B is to act as a brake for proliferative and metabolic signals (37
). However, more recent data indicate that this simple model is somewhat limited. In fibroblast, PTP1B is required for the activation of the small GTPase Ras, an enzyme that is generally associated with increased cell proliferation and motility (39
). Consistent with a positive role in signaling, PTP1B has been identified as the major tyrosine phosphatase that positively regulates the activity of endogenous Src kinase by reducing phosphorylation at Tyr-527 both in colon (40
) and in breast cancer (41
). These and other somewhat contradictory reports indicate that we are far from a system level understanding of the PTP1B network. Because the best characterized role of PTP1B is in the regulation of proliferative pathways, our in vivo
validation experiments focused on proteins that are involved in modulating the response to receptor tyrosine kinase stimulation: PLC-γ1, SHP2, Gab1, EGFR, and SHP1.
Using the substrate-trapping approach, we demonstrate a specific and direct binding of the inferred substrate proteins to the catalytic domain of PTP1B (). Consistently PTP1B overexpression down-regulates the phosphorylation of PLC-γ1, Gab1, and SHP2, after insulin or EGF stimulation (). Because it has been reported that PTP1B can act as a negative regulator of these pathways by directly de-phosphorylating the receptors (43
), we have also monitored substrate phosphorylation levels under phosphorylation conditions that are independent from growth factor receptor activity. By making use of a constitutively active, and PTP1B-insensitive form of the Src kinase, we lend further support to a direct dephosphorylation mechanism and exclude an indirect effect due to the inactivation of upstream kinases (i.e.
the receptor). The results obtained by knocking down the PTP1B concentration by an siRNA approach or by abating its enzymatic activity with a specific inhibitor are consistent with these conclusions ( and ). The observed de-phosphorylation of PLC-γ1, SHP2, and Gab1 is specific because the phosphorylation level of the adaptor protein Grb2 is found to be independent of PTP1B concentration. At the same time, increasing the concentration of TC-PTP, the closest PTP1B homologue, does not affect the phosphorylation levels of the proposed PTP1B substrates, thus confirming some level of specificity in PTP1B substrate selection ( and supplemental Fig. S5
). Direct dephosphorylation of Gab1 and PLC-γ1 by PTP1B is proved by an in vitro
protein-tyrosine phosphatase assay with purified enzyme and immunoprecipitated substrates (). PLC-γ1 and SHP2 were also in a list of putative PTP1B substrates recently reported by Mertins et al.
), whereas Gab1 is proposed and validated as a PTP1B substrate for the first time here, to our knowledge. Gab1 is a member of a family of docking/scaffolding proteins acting downstream of tyrosine kinase receptors. After EGFR stimulation, Gab1 is phosphorylated on several tyrosines that in turn work as docking sites for SH2 domain-containing signaling molecules, such as SHP2, PI3K regulatory subunit p85, Crk, Ras GTPase-activating protein, and PLC-γ1 (some of these interactions are shown in ). We asked whether the PTP1B-dependent modulation of Gab1 tyrosine phosphorylation described here could affect its ability to form signaling complexes. Among the many Gab1 binding partners, we focused on SHP2, a ubiquitously expressed protein-tyrosine phosphatase, which, in association with Gab1, has a crucial role in the receptor tyrosine kinase-dependent activation of ERK1/2 (25
). Here we have shown that PTP1B can down-regulate the formation of the SHP2-Gab1 complex by dephosphorylating Gab1 (). We also provide evidence of a physical association between Gab1, PTP1B, and the docking protein Grb2 to form a ternary complex. PTP1B has been shown to have the potential to interact directly with Grb2 via a proline-rich motif that docks into the Grb2 SH3 domains (31
). We confirmed this interaction demonstrating a considerable co-immunoprecipitation of these two proteins. More specifically we showed a constitutive interaction between PTP1B and Grb2 both in non-stimulated and EGF-stimulated cells, whereas the association of PTP1B with Gab1 was observed only after EGF induction. As mentioned before, the interaction between Grb2 and PTP1B and the functional effects on Gab1 dephosphorylation described in the present work have implications for the regulation of proliferation. The role of PTP1B in the Ras/MAP kinase pathway is controversial. Most studies have been performed in immortalized fibroblast cell lines derived from wild type and PTP1B knock-out embryos stimulated with PDGF. Although many studies point to the role of PTP1B as a growth suppressor, Haj and co-workers (10
) reported that PDGF-stimulated ERK and AKT activation is not substantially altered in PTP1B-deficient mouse embryonic fibroblasts. However, in the same cellular system under lower levels of PDGF stimulation (10–20 ng/ml), the absence of PTP1B was shown to attenuate ERK activation (39
). In our work we used a HEK293 cell line under stimulation at high EGF concentration and we observed that overexpression of PTP1B reduces ERK activation, whereas inhibition of the PTP1B enzymatic activity has an opposite effect. Taken together, these results point to a subtle control mechanism heavily dependent on the cellular and genetic context. Our results lend support to a new mechanism whereby PTP1B interferes with the association of Gab1 with SHP2 in response to EGF treatment (). The PTP1B-triggered dissociation of SHP2 mimics the Leopard syndrome mutants in SHP2 (44
), whereas favoring the membrane recruitment of p120Ras GAP, mediated by the attachment of its SH2 domain to the phosphorylated Tyr-317 of Gab1. Consistently, we observe that this rearrangement of the Gab1 complex parallels a drastic decrease in ERK activation. Given the intricacy of the network, this does not conclusively prove that dissociation of the SHP2-Gab1 complex is the only main route leading to the PTP1B-mediated ERK inactivation. In conclusion, these data suggest that PTP1B may play an important role in regulating receptor tyrosine kinase signaling pathways, by modulating the ability of Gab1 to form complexes with SH2 domain-containing proteins such as the phosphatase SHP2 and, consequently, with RasGAP and p85 PI3K.