Functional inhibition of PI3K by the βGBP molecule suppresses Ras–MAPK signalling to block cell proliferation

Abstract

The mechanisms of signal transduction from cell surface receptors to the interior of the cell are fundamental to the understanding of the role that positive and negative growth factors play in cell physiology and in human diseases. Here, we show that a functional link between phosphatidylinositol-3-OH kinase (PI3K) and Ras is suppressed by the b-galactoside binding protein (bGBP) molecule, a cytokine and a negative cell-cycle regulator. Ras-mitogen-activated protein kinase (MAPK) signalling is blocked by bGBP owing to its ability to inhibit the p110 catalytic subunit of PI3K, whose basal activity is required for Ras activation. Functional inhibition of p110 by bGBP results in downregulation of PI3K activity, suppression of Ras-GTP loading, consequent loss of MAPK activation and block of cell proliferation. This study sheds light on the molecular mechanisms whereby bGBP can control cell proliferation and, by extension, may potentially control tumorigenesis by controlling PI3K. The Ras-mitogen-activated protein kinase (MAPK) signalling pathway plays a key role in the activation dynamics of transcriptional events required for mammalian cells to enter the cell cycle. Activation of Ras requires growth factor/receptor tyrosine kinase (RTK) interaction, RTK cross-phosphorylation leading to the formation of high-affinity binding sites for SH2 domains of the Grb2 and Shc adapter proteins, the binding of the nucleotide exchange factor SOS to the SH3 domains of Grb2 and the conversion by SOS of GDP-Ras into active GTP-Ras. The ensuing signalling cascade leads to the phosphorylation of MAPK and the activation of genes programmed to initiate cell proliferation (Downward, 2003;Mitin et al., 2005 to B5 Â 10 4 receptors/cell to control cell-cycle entry and S/G 2 traverse in normal cells Mallucci, 1991, 1992). As a cytostatic factor, bGBP induces reversible S/G 2 arrest in normal cells while activating programmed cell death in cancer cells Mallucci et al, 2003). We now report that in murine embryonic fibroblasts (MEF), chosen as a paradigm of normal cells, bGBP inhibits phosphatidylinositol-3-OH kinase (PI3K) activity, also that PI3K activity is necessary for growth factor-induced activation of Ras and that basal levels are sufficient. Comparative studies with PI3K inhibitors indicate that the p110 catalytic subunit of PI3K is a prime target of bGBP-induced signalling.To determine the mode by which bGBP inhibits cell proliferation, we have investigated mechanistic events within the signalling context that lead to the phosphorylation and activation of transcription factors involved in cell-cycle entry and cell proliferation. We found (data not shown) that in MEF from secondary culture, held in G 0 by 20 nM Hu-r-bGBP and stimulated with either 10 ng/ml platelet derived growth factor (PDGF), 10 ng/ ml epidermal growth factor (EGF), 5 ng/ml fibroblast growth factor (FGF) or 10% fetal calf serum, there was no evidence of c-fos, c-jun nor, predictably, of c-myc mRNA expression indicating that no activating signals had reached the pr...

Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase

“…ERK phosphorylation was moderately enhanced by ICOS ligation in WT but not in ICOS-YF. This is consistent with the observations that PI3K can activate Ras-MAPK pathway (31,32). ICOS did not augment phosphorylation of JNK and p38 in primary CD4 ϩ blasts under our experimental settings (Fig.…”

Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer

“…On the other hand, low concentrations of EGF (31) can drive Ras activation through PI3K, which may result from the ability of PIP 3 to recruit GAP/Shp2 (32). Furthermore, PI3K inhibitors such as the cytokine β-galactosidase binding protein (βGBP) can suppress MAPK signaling (33). Through our identification of yet another feedback loop stemming from mTORC1, we now integrate this PI3K/MAPK cross-talk into the mTOR signaling network.…”

The PTEN–PI3K pathway: of feedbacks and cross-talks

“…Similarly, somatostatin receptor (Lahlou et al, 2003), as well as insulin and low doses of EGF (Wennstrom and Downward, 1999) can drive Ras activation through PI3K, which may be due to the ability of PIP 3 to recruit GAP/Shp2 in some circumstances (Yart et al, 2001;Sampaio et al, 2008). On the other hand, some PI3K inhibitors such as the cytokine b-galactosidase-binding protein (bGBP) inhibit MAPK (Wells et al, 2007). The cellular context in which PI3K signals to Ras is not yet elucidated, and further research on the role of PI3K-Ras binding, PI species production and involvement of the different PI3K isoforms is required for the proper delineation of this signaling cross-talk.…”