The same amount of protein (400-500 g) was incubated with anti-HIS TAG antibody (2 g) for at least 1 h at 4C under rotation

The same amount of protein (400-500 g) was incubated with anti-HIS TAG antibody (2 g) for at least 1 h at 4C under rotation. We also show that growth factors limit the ability of PTEN to dephosphorylate AKT. Our data emphasize the fact that PTEN acts in two unique steps of the PI3k/AKT pathway to control the expression of GLUT1 at the plasmamembrane and, further, add AKT to the list of the protein substrates of PTEN. assay using the cell homogenates as sources of both the enzyme and the substrate. For the former, we used the Endothelin Mordulator 1 PTEN protein eluted from your anti-HIS precipitates obtained from OVCAR-3 cells transfected with either the wt or Endothelin Mordulator 1 Endothelin Mordulator 1 mutant isoforms of PTEN; and for the latter we used the cell homogenate from FTC-133 cells, which do not express endogenous PTEN and constitutively express phospho-AKT at high level. After incubation of the two components, the mix was resolved by SDS-PAGE and immunoblotted with anti-phospho-AKT antibodies against the Thr308 or the Ser473 sites. The amount of PTEN in the mix was also assessed by immunoblotting with anti-HIS antibody. The results (shown in Physique 9A-9B) demonstrate that both the wt and the G129E PTEN isoforms, but not the C124S and the K128_R130del PTEN mutants, can dephosphorylate AKT at the Thr308 position, while the Ser473 appears slightly dephosphorylated only in the sample incubated with wt PTEN. Open in a separate window Physique 9 is usually a tumor suppressor gene very frequently mutated, silenced or deleted in human cancers [46]. This gene codes for any dual lipid and protein phosphatase that influences the behavior and the fate of the cell by regulating the activation of pathways that control the cell metabolism, cell survival and cell death, cell proliferation, cell migration, and genome stability [47, 48, 21]. The most common mutations involving the phosphatase domain name (coded by exon 5) of PTEN are C124S [32], G129E [49] and K128_R130del [30], among others. So far, the Y155C PTEN mutant has been described only in a glioblastoma [31]. Here we show that this ovarian malignancy cell collection OVCAR-3 also expresses this mutant isoform of PTEN. Besides the intragenic mutations, also epigenetic silencing and post-translational modifications can affect PTEN expression, stability and function [50]. Here we found that PTEN is usually epigenetically silenced through histone de-acetylation in OAW42 cells. VPA-mediated inhibition of histone de-acetylase, in fact, could rescue PTEN expression, and consequently down-regulate the AKT pathway and glucose uptake in these cells. The lipid phosphatase activity of PTEN is usually believed to play the major anti-cancer function, since the inhibition of PIP3-dependent phosphorylation of AKT impacts on a plethora of downstream pathways that control cell proliferation, apoptosis and protein synthesis besides glucose uptake [46]. Besides the lipid-phosphatase activity, PTEN possesses also a tyrosine and serine/threonine phosphatase activity [51]. Yet, the role of the protein-phosphatase activity of PTEN in malignancy is largely neglected, also because very few protein substrates involved in the malignant phenotype have been identified so far. PTEN was shown to influence cell migration by COG7 dephosphorylating FAK (Focal Adhesion Kinase) [52], chemoresistance by dephosphorylating the non-receptor Tyr kinase SRC [53], and nuclear transcription by dephosphorylating CREB (cAMP responsive-element-binding protein) [54]. More recently, it has been Endothelin Mordulator 1 reported that PTEN can dephosphorylate the insulin receptor substrate-1, thus dumping the insulin and Insulin Growth Factor signals that also impinge on glucose metabolism and cell proliferation [55]. Here we show for the first time that PTEN actually interacts with and dephosphorylates AKT. So far, the oncosuppressor function of PTEN has been attributed mainly to its lipid phosphatase activity that antagonizes the activation of the AKT pathway. Our data show that PTEN regulates this pathway also through its protein phosphatase activity. In fact, the G129E mutant that lacks the lipid phosphatase activity while retaining the protein phosphatase activity [49] could reduce the level of Trh308-phospho-AKT in the OVCAR-3 cells, which express an active PI3KC1 and an inactive Y155S PTEN mutant, and in the homogenate of FTC-133 cells, which are PTEN null and express constitutively phospho-AKT. The lowest level of phospho-AKT was achieved when the wt PTEN was ectopically expressed in OVCAR-3 cells, consistent with its dual (lipid and protein) phosphatase action in the two steps of the PI3K-AKT pathway, namely at PIP3 level and directly on the Thr308-phospho-AKT. By contrast, the C124S, lacking both the lipid and the protein phosphatase activities [32], and the K128_R130del PTEN mutants were unable to reduce the level of phospho-AKT..