Src regulates cell cycle progression and mediates cell survival through the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. This study was aimed at evaluating the potential interactions between Src and the tumor suppressor PTEN in breast cancer. PTEN dephosphorylates membrane phosphatidylinisitols (PtdIns). Upon growth factor stimulation, AKT is recruited to the activated compartments of cellular plasma membrane through the binding of its PH domain to PI3K products, PtdIns (3,4,5) triphosphates and PtdIns (3,4) bisphosphates. The effect of activated Src kinases on the ability of PTEN to dephosphorylate PtdIns was further investigated in breast cancer cells, BT-549, nucleofected with a GFP fusion protein of AKT-PH domain. The results demonstrated that active Src kinase reduces the ability of PTEN to down regulate membrane localization of AKT-PH-GFP fusion protein indicating the loss of PTEN function to dephosporylate PtdIns.
Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway