Overexpression of 14-3-3? in cancer cells activates PI3K via binding the p85 regulatory subunit.
ABSTRACT: The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3? overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3?-overexpression-mediated Akt phosphorylation, and its potential impact on breast cancer progression. We found that 14-3-3? overexpression was significantly (P=0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3? overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3? expression by small interfering RNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3? enhanced Akt phosphorylation through activation of phosphoinositide 3-kinase (PI3K). Mechanistically, 14-3-3? bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3-binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3?-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3? binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage-independent growth and enhanced stress-induced apoptosis. These findings revealed a novel mechanism by which 14-3-3? overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.
Project description:BACKGROUND:We previously identified a correlation between increased expression of the phosphoinositide 3-kinase (PI3K) regulatory subunit p85? and improved survival in human pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the impact of changes in p85? expression on response to chemotherapy and the regulation of p85? by microRNA-21 (miR-21). MATERIALS AND METHODS:PDAC tumor cells overexpressing p85? were generated by viral transduction, and the effect of p85? overexpression on sensitivity to gemcitabine was tested by MTT assay. Primary human PDAC tumors were stained for p85? and miR-21 via immunohistochemistry and in situ hybridization, respectively. Additionally, PDAC cells were treated with miR-21 mimic, and changes in p85? and phospho-AKT were assessed by Western blot. Finally, a luciferase reporter assay system was used to test direct regulation of p85? by miR-21. RESULTS:Higher p85? expression resulted in increased sensitivity to gemcitabine (P < 0.01), which correlated with decreased PI3K-AKT activation. Human tumors demonstrated an inverse correlation between miR-21 and p85? expression levels (r = -0.353, P < 0.001). In vitro, overexpression of miR-21 resulted in decreased levels of p85? and increased phosphorylation of AKT. Luciferase reporter assays confirmed the direct regulation of p85? by miR-21 (P < 0.01). CONCLUSIONS:Our results demonstrate that p85? expression is a determinant of chemosensitivity in PDAC. Additionally, we provide novel evidence that miR-21 can influence PI3K-AKT signaling via its direct regulation of p85?. These data provide insight into potential mechanisms for the known relationship between increased p85? expression and improved survival in PDAC.
Project description:Recent studies have shown that hyperinsulinemia may increase the cancer risk. Moreover, many tumors demonstrate an increased activation of IR signaling pathways. Phosphatidylinositol 3-kinase (PI3K) is necessary for insulin action. In epithelial cells, which do not express GLUT4 and gluconeogenic enzymes, insulin-mediated PI3K activation regulates cell survival, growth, and motility. Although the involvement of the regulatory subunit of PI3K (p85? (PI3K)) in insulin signal transduction has been extensively studied, the function of its N-terminus remains elusive. It has been identified as a serine (S83) in the p85? (PI3K) that is phosphorylated by protein kinase A (PKA). To determine the molecular mechanism linking PKA to insulin-mediated PI3K activation, we used p85? (PI3K) mutated forms to prevent phosphorylation (p85A) or to mimic the phosphorylated residue (p85D). We demonstrated that phosphorylation of p85? (PI3K)S83 modulates the formation of the p85? (PI3K)/IRS-1 complex and its subcellular localization influencing the kinetics of the insulin signaling both on MAPK-ERK and AKT pathways. Furthermore, the p85? (PI3K)S83 phosphorylation plays a central role in the control of insulin-mediated cell proliferation, cell migration, and adhesion. This study highlights the p85? (PI3K)S83 role as a key regulator of cell proliferation and motility induced by insulin in MCF-7 cells breast cancer model.
Project description:It has been reported that p21-activated kinase 4 (PAK4) is amplified in pancreatic cancer tissue. PAK4 is a member of the PAK family of serine/threonine kinases, which act as effectors for several small GTPases, and has been specifically identified to function downstream of HGF-mediated c-Met activation in a PI3K dependent manner. However, the functionality of PAK4 in pancreatic cancer and the contribution made by HGF signalling to pancreatic cancer cell motility remain to be elucidated. We now find that elevated PAK4 expression is coincident with increased expression levels of c-Met and the p85? subunit of PI3K. Furthermore, we demonstrate that pancreatic cancer cells have a specific motility response to HGF both in 2D and 3D physiomimetic organotypic assays; which can be suppressed by inhibition of PI3K. Significantly, we report a specific interaction between PAK4 and p85? and find that PAK4 deficient cells exhibit a reduction in Akt phosphorylation downstream of HGF signalling. These results implicate a novel role for PAK4 within the PI3K pathway via interaction with p85?. Thus, PAK4 could be an essential player in PDAC progression representing an interesting therapeutic opportunity.
Project description:Chemoresistance in gastric cancer is the leading cause of tumor recurrence and poses a substantial therapeutic challenge. The stem cell biomarker CD133 has been implicated in drug resistance of tumor-initiating cells in a number of cancers including gastric cancer. Therefore, we investigated the molecular mechanism of CD133-associated multidrug resistance in gastric cancer cells. Using CD133 overexpressing and knockdown gastric cancer cell lines, we demonstrated that loss of CD133 significantly increased the growth inhibition of chemotherapeutic agents; whereas, overexpression significantly reduced growth inhibition. Furthermore, CD133 knockdown significantly reduced the enzymatic activity of phosphatidylinositol-3 kinase (PI3K) and the expression of P-glycoprotein (P-gp), B-cell lymphoma 2 (BCL2), and phosphorylated-protein kinase B (p-AKT), but elevated the expression of BCL2 associated X (BAX). Conversely, overexpression of CD133 significantly increased PI3K enzymatic activity, expression of P-gp, BCL2, and p-AKT, and decreased BAX expression. The PI3K/AKT inhibitor LY294002 mirrored the effects of loss of CD133; whereas, the PI3K/AKT activator epidermal growth factor reproduced the effects of CD133 overexpression. To identify the interaction between CD133 and PI3K, we used site-directed mutagenesis to mutate individual tyrosine residues of CD133. We found that binding between CD133 and p85, the regulatory subunit of PI3K, was significantly reduced when tyrosine 852 was mutated. In summary, we have demonstrated that CD133 activates the PI3K/AKT signal transduction pathway through direct interaction with PI3K-p85, resulting in multidrug resistance of gastric cancer cells. These results suggest that the interaction between CD133 and PI3K-p85 may offer a novel therapeutic target in multidrug resistant gastric cancer.
Project description:The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110beta isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.
Project description:BACKGROUND: The ?-isoform of the Type 1A Phosphoinositide 3-kinases (PI3K?) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85?, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. RESULTS: We have assessed whether oncogenic mutants of PI3K?, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3K?, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3K? with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3K?. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3K? affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3K?. CONCLUSIONS: Phosphorylation of p85? S608 is not a significant regulator of wild-type or oncogenic PI3K? lipid kinase activity.
Project description:The I?B kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability to regulate NF-?B, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition.
Project description:Phosphoinositide 3-kinase (PI3K) activity is important for regulating cell growth, survival, and motility. We report here the identification of bromodomain-containing protein 7 (BRD7) as a p85?-interacting protein that negatively regulates PI3K signaling. BRD7 binds to the inter-SH2 (iSH2) domain of p85 through an evolutionarily conserved region located at the C terminus of BRD7. Via this interaction, BRD7 facilitates nuclear translocation of p85?. The BRD7-dependent depletion of p85 from the cytosol impairs formation of p85/p110 complexes in the cytosol, leading to a decrease in p110 proteins and in PI3K pathway signaling. In contrast, silencing of endogenous BRD7 expression by RNAi increases the steady-state level of p110 proteins and enhances Akt phosphorylation after stimulation. These data suggest that BRD7 and p110 compete for the interaction to p85. The unbound p110 protein is unstable, leading to the attenuation of PI3K activity, which suggests how BRD7 could function as a tumor suppressor.
Project description:The biological significance of a known normal and cancer stem cell marker CD133 remains elusive. We now demonstrate that the phosphorylation of tyrosine-828 residue in CD133 C-terminal cytoplasmic domain mediates direct interaction between CD133 and phosphoinositide 3-kinase (PI3K) 85 kDa regulatory subunit (p85), resulting in preferential activation of PI3K/protein kinase B (Akt) pathway in glioma stem cell (GSC) relative to matched nonstem cell. CD133 knockdown potently inhibits the activity of PI3K/Akt pathway with an accompanying reduction in the self-renewal and tumorigenicity of GSC. The inhibitory effects of CD133 knockdown could be completely rescued by expression of WT CD133, but not its p85-binding deficient Y828F mutant. Analysis of glioma samples reveals that CD133 Y828 phosphorylation level is correlated with histopathological grade and overlaps with Akt activation. Our results identify the CD133/PI3K/Akt signaling axis, exploring the fundamental role of CD133 in glioma stem cell behavior.
Project description:Phosphoinositide 3-kinase (PI3K) is an important therapeutic target. Mutations in PIK3CA, which encodes p110?, the catalytic subunit of PI3K, occur in endometrioid endometrial cancers (EEC) and nonendometrioid endometrial cancers (NEEC). The goal of this study was to determine whether PIK3R1, which encodes p85?, the inhibitory subunit of PI3K, is mutated in endometrial carcinoma. We carried out exonic sequencing of PIK3R1 from 42 EECs and 66 NEECs. The pattern of PIK3R1 mutations was compared with the patterns of PIK3CA, PTEN, and KRAS mutations. The biochemical effect of seven PIK3R1 mutations was examined by stable expression in U2OS cells, followed by coimmunoprecipitation analysis of p110?, and Western blotting of phospho-AKT(Ser473) (p-AKT(Ser473)). We found that PIK3R1 was somatically mutated in 43% of EECs and 12% of NEECs. The majority of mutations (93.3%) were localized to the p85?-nSH2 and -iSH2 domains. Several mutations were recurrent. PIK3R1 mutations were significantly (P = 0.0015) more frequent in PIK3CA-wild type EECs (70%) than in PIK3CA mutant EECs (18%). Introduction of wild-type p85? into U2OS cells reduced the level of p-AKT(Ser473) compared with the vector control. Five p85? mutants, p85?delH450-E451, p85?delK459, p85?delY463-L466, p85?delR574-T576, and the p85?N564D positive control, were shown to bind p110? and led to increased levels of p-AKT(Ser473). The p85?R348X and p85?K511VfsX2 mutants did not bind p110? and showed no appreciable change in p-AKT(Ser473) levels. In conclusion, our study has revealed a new mode of PI3K alteration in primary endometrial tumors and warrants future studies to determine whether PIK3R1 mutations correlate with clinical outcome to targeted therapies directed against the PI3K pathway in EEC and NEEC.