These results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.

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6 Corresponding author p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110␣ is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.

Both the Raf-MAP kinase and PI 3-kinase-PKB/Akt pathways are activated by Ras, but only the PI 3-kinase-PKB/Akt pathway is activated by R-Ras. PI 3-kinase, and downstream targets such as PKB/Akt, are likely to be essential mediators of transformation induced by R-Ras. PI 3-kinase, as well as Raf, is thus implicated also in Ras transformation.