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...

Src homology 2 (SH2) domains bind specifically to tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes. A protein domain was identified that bound specifically to the tyrosine-phosphorylated form of its target protein but differs from known SH2 sequences. Phosphotyrosine-binding (PTB) domains were found in two proteins: SHC, a protein implicated in signaling through Ras; and SCK, encoded by a previously uncharacterized gene. The PTB domain of SHC specifically bound to a tyrosine-phosphorylated 145-kilodalton protein. PTB domains are an alternative to SH2 domains for specifically recruiting tyrosine-phosphorylated proteins into signaling complexes and are likely to take part in signaling by many growth factors.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3 phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110*, which can stimulate Akt activity. We used purified p110* protein to generate a series of 3 phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P 2 ) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P 3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P 2 or after enzymatic conversion of PI3,4,5P 3 into PI3,4P 2 with the signaling inositol polyphosphate 5 phosphatase SIP. We show that PI3,4P 2 -mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P 2 . Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.A number of studies implicate phosphatidylinositol (PI) 3-kinase in the regulation of cell growth and transformation (for reviews, see references 4, 17, and 34). Treatment of cells with mitogenic stimuli as well as oncogenic transformation causes a rapid and transient increase in the level of products of PI 3-kinase, PI3,4-bisphosphate (P 2 ), and PI3,4,5-triphosphate (P 3 ). These 3Ј phosphorylated inositol phospholipids are believed to represent a novel class of second messengers; however, their function with respect to PI 3-kinase-induced signaling remains largely unknown. Certain isotypes of protein kinase C have been shown to be activated by PI3,4P 2 and PI3,4,5P 3 in vitro (28, 37), but it remains unclear whether they are effectors of PI 3-kinase in vivo, and in which way the activation of this pathway contributes to PI 3-kinase-mediated signaling. PI3,4,5P 3 was shown to interact with Src homology 2 (SH2) domains and to modulate the association of PI 3-kinase with tyrosine-phosphorylated molecules (29). PI3,4P 2 and PI3,4,5P 3 can stimulate pleckstrin phosphorylation in permeabilized platelets (36, 40). The protein kinase Akt (also known as Rac-protein kinase or protein kinase B) has been suggested to be stimulated by PI3P (10). However, the effect of PI3,4P 2 and PI3,4,5P 3 , which are generated in response to mitogenic stimuli, was not investigated.Studies of PI 3-kinase were facilitated by the recent development of constitutively active PI 3-kinase molecules, which can induce ...

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.

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...