Conditional Inhibition of the Mitogen-activated Protein Kinase Cascade by Wortmannin

Abstract

Phosphoinositide (PI) 3-kinase and the mitogen-activated protein (MAP) kinase cascades are activated by many of the same ligands. Several groups have reported involvement of PI 3-kinase in the activation of Erk1 and Erk2, whereas many other groups have shown that activation of Erk1 and Erk2 is not sensitive to inhibitors of PI 3-kinase such as wortmannin. Here we show that wortmannin inhibition of the MAP kinase pathway is cell type-and ligand-specific. Wortmannin blocks platelet-derived growth factor (PDGF)-dependent activation of Raf-1 and the MAP kinase cascade in Chinese hamster ovary cells, which have few PDGF receptors, but has no significant effect on Erk activation in Swiss 3T3 cells, which have high levels of PDGF receptors. However, wortmannin blocks activation of Erk proteins if Swiss 3T3 cells are stimulated with lower, physiological levels of PDGF. These results suggest that PI 3-kinase is in an efficient pathway for activation of MAP kinase, but that MAP kinase can be stimulated by a redundant pathway when a large number of receptors are activated. We present evidence that a protein kinase C family member downstream of phospholipase C␥ is involved in the redundant pathway. PI1 3-kinase has been implicated as being involved in the signal transduction of virtually all growth factors studied and in the transformation of cells by several oncoproteins (1, 2). Activation of PI 3-kinase with growth factors results in the appearance of the lipid products of this enzyme, PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 , within seconds to minutes. There is also a correlation between the elevation in PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 levels and cell transformation. These correlations have suggested an important role for PI 3-kinase in signal transduction pathways leading to cell growth and transformation. Indeed, the catalytic subunit of PI 3-kinase (p110␣) has recently been identified as a retroviral oncogene (3). Overexpression of p110␣ in chick embryo fibroblasts results in constitutive elevation of PtdIns-3,4-P 2 and PtdIns-3,4,5-P 3 and cell transformation.The MAP kinase pathway is another key component in the transduction of signals leading to growth and transformation. This pathway consists of a linear cascade of the protein kinases Raf, MEK, and MAP kinase/Erk; like PI 3-kinase, Erk1 and Erk2 are acutely activated by growth factors and are found constitutively activated in many transformed cell lines. The Erk proteins are phosphorylated and activated by the dual specificity kinase MEK (MAP kinase/Erk kinase), which is phosphorylated and activated by the serine/threonine kinase Raf. Raf is recruited to the membrane of activated cells by direct binding to Ras-GTP. This recruitment by activated Ras is necessary but not sufficient for full activation of Raf (4); therefore, other factors that are necessary for activation of the MAP kinase pathway may feed into the pathway at Raf. A search for other sources that feed into the MAP kinase pathway has yielded several candidates including Src, members of the PKC family, ...

Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase

“…Several authors have reported that PI3K inhibitors abrogate p42\p44 MAPK activation in response to growth-factor tyrosine kinase and G i -coupled receptors [1,[5][6][7]. We have shown here that PI3K inhibitors (LY294002, 50 µM ; wortmannin, 50 nM) markedly decreased the activation of p42\p44 MAPK by PDGF (Table 1, Figure 5).…”

“…GTP GDP exchange in Ras promotes activation of the serine\ threonine protein kinase c-Raf, MAPK kinase (MEK-1) and p42\p44 MAPK. Duckworth and Cantley [1] have also demonstrated an essential role for phosphoinositide 3-kinase (PI3K) in platelet-derived growth-factor-receptor (PDGFR)-dependent regulation of this pathway in Swiss 3T3 fibroblasts.…”

S338 Phosphorylation of Raf-1 Is Independent of Phosphatidylinositol 3-Kinase and Pak3

“…When low concentrations of growth factors are used, or low-density receptors are stimulated, P13-K appears to play a permissive role in Ras/ERK signaling (16,56). By contrast, at higher growth factor concentrations or when higher-density receptors are activated, P13-K function appears not to be required (16,56). We now show that even at high growth factor concentrations in COS cells, the P13-K inhibitors suppressed Ras activation, but only at later times (Fig.…”

“…However, it is now clear that Ras also regulates the activity of a number of other signaling pathways through activation of proteins such as phosphatidylinositol 3-kinase (Pl3-K) and RalGDS (4,55). There is much interest in the cross talk that exists between these different, parallel Ras pathways, and in some but not all cell types, inhibition of Pl3-K that leads to suppression of ERK has been shown (8,16,24,56). In different studies, Pl3-K has been shown to regulate ERK activation at the level of Ras or at the level of Raf-1 (7,8,27,56).…”

Cutting Edge: Extracellular Signal-Regulated Kinases 1/2 Function as Integrators of TCR Signal Strength

“…It has previously been shown in mouse lymph node cells that anti-CD3 stimulation with concurrent inhibition of PI-3K activity leads to diminished activity of ERK-2 (13). Furthermore, in nonimmune cells, ERK activity is sensitive to PI-3K inhibition when relatively few growth factor receptors are engaged, but more resistant when a larger number of receptors are activated (14). Because IL-2 signaling transduces the PI-3K pathway (15,16), we compared the ability to induce ERK activation in effector PBT taken directly from culture with IL-2 vs those removed from IL-2 for 16 h before stimulation through the TCR.…”