Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of p110* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinasedependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.Phosphatidylinositol (PI) 3-kinase activity has been implicated in the regulation of a number of different cellular responses, including the regulation of cell growth. Oncogenic transformation or the stimulation of cells with growth factors results in an increased level in the phospholipid products of PI 3-kinase (for reviews, see references 4, 23, and 50). Mutants of platelet-derived growth factor (PDGF) receptor or certain oncogenes which fail to activate PI 3-kinase are deficient in triggering either mitogenic responses or oncogenic transformation, respectively. These data suggest that PI 3-kinase is an important mediator of signaling events that regulate cell growth and cellular transformation.PI 3-kinase is a heterodimeric complex consisting of 85-and 110-kDa subunits (p85 and p110) (23). The p85 subunit consists of multiple domains including a Src homology 3 (SH3) domain, a breakpoint cluster region domain, and two SH2 domains. The two SH2 domains bind tyrosine-phosphorylated receptors and in this manner recruit the p85-p110 complex to activated receptors. The two SH2 domains are separated by the inter-SH2 (iSH2) region. The iSH2 domain mediates the interaction of p85 with p110, and this interaction is required for the enzymatic activity of p110 (28) (Fig. 1). It is possible that the role of p85 is to target p110 to the membrane, where its lipid substrates reside (23).PI 3-kinase has been implicated in the regulation of many other cellular processes, including the reorganization of the actin cytoskeleton (24,30,40,59), receptor internalization (21), histamine secretion (63), neutrophil activation (56), platelet activation (64), cell migration (31), glucose transport (41), and vesicular sorting (48). PI 3-kinase was implicated in the...

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.Phosphatidylinositol (PI) 3-kinase has been shown to mediate signaling induced by numerous growth factors and tumor antigens. The intracellular levels of the phospholipid products of PI 3-kinase increase in response to stimulation with growth factors or after oncogenic transformation (for reviews, see references 10,11,33,76,80). PI 3-kinase signaling appears to be required for a number of mitogens during the G 1 -to-Sphase transition of the cell cycle (63). Recently, it was demonstrated that PI 3-kinase regulates cell survival in response to various apoptotic stimuli (21, 49).PI 3-kinase is a heterodimeric complex consisting of an 85-kDa regulatory subunit, p85, and a 110-kDa catalytic subunit, p110 (11, 33). The p85 subunit contains two Src homology 2 (SH2) domains, which bind to tyrosine-phosphorylated receptors after stimulation of cells with growth factors and in this manner recruit the p85-p110 complex to the cell membrane. The region between the two SH2 domains of p85, the iSH2 region, mediates the association with p110, and this interaction is required for the enzymatic activity of p110 (37). Based on this observation we generated a chimeric molecule, p110*, in which the iSH2 region of p85 was covalently linked to its binding site at the p110 N terminus by using a flexible hinge region (30). p110* is a constitutively active PI 3-kinase which can activate signaling pathways independent of growth factor stimulation.The generation of constitutively active PI 3-kinase molecules has greatly facilitated the analysis of signa...

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