Requirement for Phosphatidylinositol 3-Kinase in Epidermal Growth Factor-Induced AP-1 Transactivation and Transformation in JB6 P⁺ Cells

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) is an important enzyme associated with a variety of receptors or proteintyrosine kinases and acts as a direct biochemical link between a novel phosphatidylinositol pathway and a number of receptor proteins, including the receptors for insulin or platelet-derived growth factor. This enzyme is a heterodimer of a 110-kDa (P110) catalytic subunit and an 85-kDa (P85) regulatory subunit (5). It can phosphorylate phosphatidylinositol (Ptdins), Ptdins(4) phosphate [Ptdins(4)P], or Ptdins(4,5) bisphosphate [Ptdins(4,5)P2] to produce Ptdins(3)P, Ptdins(3,4)P2, or Ptdins (3,4,5) trisphosphate [Ptdins(3,4,5)P3], respectively (2, 5, 52). Insulin or growth factor stimulation of the associated tyrosine kinase results in phosphorylation of the P85 subunit of PI 3-kinase. This phosphorylation is important for activation of PI 3-kinase (6).Several studies suggested that the PI 3-kinase products Ptdins(3,4)P2 and Ptdins(3,4,5)P3 are important regulators of cell proliferation (4, 21, 23). The introduction of the N-terminal SH 2 domain of the P85 subunit of PI 3-kinase into cells abrogates insulin-or insulin-like growth factor I (IGF-I)-stimulated DNA synthesis and prevents insulin stimulation of c-fos protein expression (21, 23). Other studies have shown that Ptdins(3,4)P2 and Ptdins(3,4,5)P3 levels are elevated in cells transformed by v-abl, v-src, and polyomavirus middle T, and decreased levels of these lipids correlate with impaired cell transformation by mutated forms of these oncogenes (4,16,47). Recently, it was reported that insulin could activate the Ras-Raf-mitogen-activated protein (MAP) kinase pathway by interacting with and activating its receptors (45, 53). Hu et al. suggest that activation of this Ras-MAP kinase pathway is critical for the effect of insulin on mitogenesis and c-fos expression (21). Others found that neither insulin nor phorbol ester regulation of phosphoenolpyruvate carboxykinase gene expression requires activation of the Ras-MAP kinase pathway but that PI 3-kinase is required in this event (17, 54). In contrast, Sakaue et al. demonstrated that neither the Ras-MAP kinase cascade nor PI 3-kinase may be required for insulinstimulated glycogen synthase activation in CHO cell lines (32). It is well known that epidermal growth factor (EGF) can induce a high level of AP-1 activity and cell transformation as well as PI 3-kinase activity. Cell transformation is a complex process and much different from cell mitogenesis; in some cases, these two events are dissociated (7,20,29,41,51). Currently, little is known regarding whether PI 3-kinase and its products are involved in EGF-induced signal transduction to the transcriptional machinery of the nucleus and cell transformation. We used the well-characterized mouse epidermal JB6 P ϩ (tumor promotion-sensitive) cells and addressed a novel function of PI 3-kinase in EGF-induced AP-1 transactivation and cell transformation. MATERIALS AND METHODSPlasmids and reagents. The AP-1 luciferase reporter plasmid (Col-Luc) and CMV-n...

The Prolyl Isomerase Pin1 Enhances HER-2 Expression and Cellular Transformation via Its Interaction with Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase 1

“…In addition, TPA and EGF are associated with a neoplastic cell transformation in JB6 Cl41 cells (46)(47)(48). Our results show that the overexpression of Pin1 induced a neoplastic cell transformation in JB6 Cl41 cells, and EGF enhanced the Pin1-induced cell transformation (Fig.…”

Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes

“…Our results showing that a TGFa initiated cell line is especially sensitive to low concentrations of LY294002 are also noteworthy in light of studies with neutralizing antibodies demonstrating that PI 3-kinase is required for signaling by the EGF receptor (Auger et al, 1989;Jackson et al, 1992;Roche et al, 1994). Furthermore, studies with a dominant negative p85 construct (encoding a mutant version of the regulatory subunit of PI 3-kinase) and chemical inhibitors such as we use here, have showed that PI 3-kinase is required for EGF receptor mediated transformation of mouse epidermal cells in vitro (Huang et al, 1996).…”

Recruitment of insulin receptor substrate-1 and activation of NF-κB essential for midkine growth signaling through anaplastic lymphoma kinase

“…These results suggest that ALK can drive NF-kB activation, and that activation is increased with the presence of IRS-1. In addition, the lack of activation of AP-1, SRE and SRF promoter/reporter constructs indicates that the ALK/ IRS-1-mediated MAPK and PI3-kinase activity does not cause a global increase of transcription of all mitogenesis-and survival-related pathways Deng and Karin, 1994;Minden et al, 1994;Huang et al, 1996) but instead a relatively selective activation of NF-kB in this cellular context. The significance of this activation is supported by the effect of the knockdown of the p65 subunit of NF-kB, which disrupts the transformed phenotype of the 32D/ IRS-1/ALK cells (Figure 7).…”

“…Furthermore, only cells containing both ALK and IRS-1 showed this NF-kB activity (Figure 6b not show any induction in any of the cell lines, although a small but inconsistent activation of the AP-1 promoter/reporter was seen with the 32D/IRS-1 ALK cells (Figure 6a). This lack of AP-1 activity was particularly surprising as control experiments with 32D cells showed that expression of pFC-MEKK, a constitutively active MEKK, induced activation of the AP-1 promoter/ reporter by eightfold (not shown) and AP-1 had been shown by others to be activated by MAPK (Deng and Karin, 1994;Minden et al, 1994) and PI3-kinase (Huang et al, 1996) signaling.…”