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.

Cell migration represents an important cellular response that utilizes cytoskeletal reorganization as its driving force. Here, we describe a new signaling cascade linking PDGF receptor stimulation to actin rearrangements and cell migration. We demonstrate that PDGF activates Cdc42 and its downstream effector N-WASP to mediate filopodia formation, actin stress fiber disassembly, and a reduction in focal adhesion complexes. Induction of the Cdc42 pathway is independent of phosphoinositide 3-kinase (PI3K) enzymatic activity, but it is dependent on the p85α regulatory subunit of PI3K. Finally, data are provided showing that activation of this pathway is required for PDGF-induced cell migration on collagen. These observations show the essential role of the PI3K regulatory subunit p85α in controlling PDGF receptor–induced cytoskeletal changes and cell migration, illustrating a novel signaling pathway that links receptor stimulation at the cell membrane with actin dynamics.

Hypoxia is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3-4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.

Alterations in the cell division:cell death ratio induce multiple autoimmune and transformation processes. Phosphoinositide 3-kinase (PI3K) controls cell division and cell death in vitro, but its effect on the function of the cellular immune system and on tumor formation in mammals is poorly characterized. Here we show that transgenic mice expressing in T lymphocytes an active form of PI3K derived from a thymic lymphoma, p65(PI3K), developed an infiltrating lymphoproliferative disorder and autoimmune renal disease with an increased number of T lymphocytes exhibiting a memory phenotype and reduced apoptosis. This pathology was strikingly similar to that described in mice exhibiting heterozygous loss of the tumor suppressor PTEN, a lipid and protein phosphatase. We show that overexpression of PTEN selectively blocks p65(PI3K)-induced 3T3 fibroblast transformation. Moreover, the early development of T cell lymphomas in p65(PI3K) Tg p53(-/-) mice indicated that PI3K contributes to tumor development. These observations demonstrate that constitutive activation of PI3K extends T cell survival in vivo, affects T cell homeostasis, and contributes to tumor generation, supporting a model in which selective increases in one type of PTEN substrate, the PI3K-derived lipid products, induce tumorigenesis. PI3K thus emerges as a potential target in autoimmune disease and cancer therapy.

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Rasinduced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.Phosphoinositide 3-kinases (PI3K) 1 are enzymes that transfer phosphate to position 3 of the phosphoinositide ring, regulating a variety of cell responses including survival, division, and transformation. PI3Ks are divided into three subclasses based on their primary structure and substrate specificity, but only the class I enzymes generate phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (3-poly-PtdIns) products in vivo. Basal levels of these lipids are very low in quiescent cells but increase rapidly and transiently following growth factor receptor (GFR) stimulation (for a review, see Refs. 1-4). 3-Poly-PtdIns recruits pleckstrin homology domain-containing proteins such as phosphoinositide-dependent kinase-1 and protein kinase B (PKB), which mediate PI3K signal propagation (5-8).Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, of which there are several isoforms (1, 2, 9 -14). The prototypic p85 regulatory subunit has an SH3 domain, a BcR homology domain (BH) flanked by proline-rich sequences, and two SH2 domains separated by the so-called inter-SH2 domain, to which p110 binds (15-17). The p110 catalytic subunit contains a p85-interacting region, a Ras-binding domain, a region homologous to PI4K and the PI3K catalytic domain (4, 15). The p85 subunit protects p110 from degradation and inhibits its enzymatic activity in quiescent cells (18). When cells are stimulated by receptors with intrinsic Tyr kinase activity, such as platelet-derived growth factor receptor (PDGFR), p85 mediates p110 translocation to the cell membrane, where PI3K substrates are found (11). In addition to mediating PI3K translocation, p85 appears to transmit an activating conformational change to p110 (19 -21), because binding to p85 of Tyr-phosphorylated peptides representing the activated PDGFR increases p110 enzymatic activity (16,22). Other receptors activate PI3K by stimulating cytosolic Tyr ki...

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Heart failure (HF) is a major health and economic burden in developed countries. It has been proposed that the pathogenesis of HF may involve the action of mitochondria. We evaluate three different mouse models of HF: tachycardiomyopathy, HF with preserved left ventricular (LV) ejection fraction (LVEF), and LV myocardial ischemia and hypertrophy. Regardless of whether LVEF is preserved, our results indicate that the three models share common features: an increase in mitochondrial reactive oxygen species followed by ultrastructural alterations in the mitochondrial cristae and loss of mitochondrial integrity that lead to cardiomyocyte death. We show that the ablation of the mitochondrial protease OMA1 averts cardiomyocyte death in all three murine HF models, and thus loss of OMA1 plays a direct role in cardiomyocyte protection. This finding identifies OMA1 as a potential target for preventing the progression of myocardial damage in HF associated with a variety of etiologies.

The serine/threonine kinase p70 S6 kinase (p70S6K) phosphorylates the 40 S ribosomal protein S6, modulating the translation of an mRNA subset that encodes ribosomal proteins and translation elongation factors. p70S6K is activated in response to mitogenic stimuli and is required for progression through the G 1 phase of the cell cycle and for cell growth. Activation of p70S6K is regulated by phosphorylation of seven different residues distributed throughout the protein, a subset of which depends on the activity of p85/p110 phosphatidylinositol 3-kinase (PI3K); in fact, the phosphorylation status of Thr 229 and Thr 389 is intimately linked to PI3K activity. In the full-length enzyme, however, these sites are also acutely sensitive to the action of FKBP 12-rapamycin-associated protein (FRAP). The mechanism by which PI3K and FRAP cooperate to induce p70S6K activation remains unclear. Here we show that the p85 regulatory subunit of PI3K also controls p70S6K activation by mediating formation of a ternary complex with p70S6K and FRAP. The p85 C-terminal SH2 domain is responsible for p85 coupling to p70S6K and FRAP, because deletion of the C-terminal SH2 domain inhibits complex formation and impairs p70S6K activation by PI3K. Formation of this complex is not required for activation of a FRAP-independent form of p70S6K, however, underscoring the role of p85 in regulating FRAPdependent p70S6K activation. These studies thus show that, in addition to the contribution of PI3K activity, the p85 regulatory subunit plays a critical role in p70S6K activation.