Protein tyrosine phosphatase (PTP) 1B is an abundant, cytoplasmic enzyme that plays a major role in down-regulating insulin and leptin signaling (1
). PTP1B binds to and dephosphorylates the insulin receptor, thus terminating signals from this receptor tyrosine kinase (RTK). Deletion of the Ptp1b
gene in mice causes hypersensitivity to insulin, and is associated with marked increases in tyrosine phosphorylation of the insulin receptor and its targets (2
). PTP1B may also inhibit signaling from other RTKs such as the PDGF and HGF receptors (4
). Consistent with its role as an inhibitor of RTKs, overexpression of PTP1B in fibroblasts inhibits transformation by oncogenes that increase tyrosine phosphorylation, including ErbB2, Src, Bcr-Abl, and Crk, and also by Ras (8
). In Src or Crk transformed cells, overexpression of PTP1B is associated with loss of tyrosine phosphorylation of key signaling proteins such as Crk and p130Cas
The above results, coupled with effects of PTP1B on the insulin receptor, gave rise to the idea that the main role of PTP1B is to act as a brake to proliferative and metabolic signals. However, recent data indicate that this idea is too limited. In fibroblasts, PTP1B is required for the activation of the small GTPases Ras (12
) and Rac (13
), enzymes that are generally associated with increased cell proliferation and motility. In addition, PTP1B has also been shown to activate Src by dephosphorylating the inhibitory Y527 site in the C-terminus of this kinase (13
). Thus, at endogenous levels of expression, PTP1B has certain pro-growth properties, in marked contrast to its aforementioned ability to revert transformation by various oncogenes when overexpressed. In vivo
mice resist transformation by the ErbB2 (a.k.a.
Neu/HER2) oncogene, and transgenic overexpression of PTP1B in mammary cells is oncogenic (17
). Moreover, in a significant fraction of human clinical samples of ErbB2-positive breast cancer, amplification of chromosome 20q13 (containing the PTP1B gene) has been noted, with increased expression of PTP1B (19
). These findings imply that this enzyme plays a positive role in growth signaling in at least some tissues, and that PTP1B might therefore serve as a therapeutic target in certain malignancies.
How does PTP1B contribute to oncogenesis in mammary cells? PTP1B is known to up-regulate two growth-promoting pathways: it activates Src and deactivates p62Dok
, an inhibitor of the Ras/MAPK pathway (21
). Regarding these substrates, it is at least theoretically plausible that the reported in vivo
effects of PTP1B loss on ErbB2-driven breast cancer can be explained in terms of failure to activate Src. However, Kaminski et al.
have recently reported that Src activity is dispensable for ErbB2-driven carcinogenesis (22
). Another PTP1B substrate - p62Dok
- represents an alternate explanation for the positive effects of PTP1B on transformation. When tyrosine phosphorylated, this protein complexes with, and activates, p120RasGAP
, leading to decreased Ras and MAPK activity (23
). Loss of PTP1B leads to hyperphosphorylation of p62Dok
, with consequent inactivation of Ras and its downstream effectors. However, tissue samples from ErbB2/Ptp1b−/−
mice gave inconsistent results regarding the tyrosine phosphorylation status of p62Dok
). For these reasons, whether the observed effects of PTP1B on ErbB2-driven carcinogenesis are due to the interactions of PTP1B with Src, p62Dok
, or unidentified substrates is not known.
In this paper, we sought to clarify the molecular mechanism(s) by which PTP1B contributes to ErbB2 signaling in breast epithelial cells. We used a 3D in vitro model to recapitulate the architectural elements of breast acinar development, while retaining the ability to manipulate and analyze the pathways that underlie the effects of PTP1B on ErbB2 signaling. Consistent with its proposed role in oncogenic signaling, we found that activation of ErbB2, but not ErbB1, increased PTP1B expression, and that siRNA-induced reduction in PTP1B expression or inhibition of PTP1B activity by small molecule inhibitors impeded the ability of activated ErbB2 to transform these cells and to activate Src and its associated downstream signaling targets. In addition, we found that the suppressive effects of PTP1B loss could be bypassed by expression of mutationally activated Src. Likewise, we found that overexpression of PTP1B in breast epithelial cells distorted normal acinar morphology, causing unchecked proliferation, and loss of polarity. These effects were associated with Src activation and required the presence of protein phosphatase activity and the Src-binding motif in the C-terminus of PTP1B. These results support a model in which PTP1B, by activating Src, cooperates with ErbB2 in transforming mammary epithelial cells.