Direct association of p110 beta phosphatidylinositol 3-kinase with p85 is mediated by an N-terminal fragment of p110 beta.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme of 85-kDa (p85) and 110-kDa (p110) subunits implicated in mitogenic signal transduction by virtue of its activation in cells transformed by diverse viral oncoproteins and treated with various growth factors. We have identified a domain in p110 that mediates association with p85 in vitro and in intact cells. A glutathione S-transferase fusion protein containing the N-terminal 171 amino-acids of p110 beta bound to free p85 in cell lysates. This fusion protein also bound directly to p85 immobilized on nitrocellulose filters. An epitope-tagged fragment containing amino acids 31 to 150 of p110 beta associated with p85 upon expression in intact cells. Expression of either an N-terminal fragment of p110 beta or the p85 inter-SH2 domain, which mediates association with p110, reduced the association of endogenous PI 3-kinase activity with the activated platelet-derived growth factor receptor in intact cells. Hence, these defined regions of p85 and p110 mediate the interaction between the two subunits of PI 3-kinase.     

Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.     

cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor.

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.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Epidermal growth factor-induced phosphatidylinositol 3-kinase activation and DNA synthesis. Identification of Grb2-associated binder 2 as the major mediator in rat hepatocytes

In previous work we showed that the phosphatidylinositol 3-kinase (PI3-kinase), not the mitogen-activated protein kinase, pathway is necessary and sufficient to account for insulin- and epidermal growth factor (EGF)-induced DNA synthesis in rat hepatocytes. Here, using a dominant-negative p85, we confirmed the key role of EGF-induced PI3-kinase activation and sought to identify the mechanism by which this is effected. Our results show that EGF activates PI3-kinase with a time course similar to that of the association of p85 with three principal phosphotyrosine proteins (i. e. PY180, PY105, and PY52). We demonstrated that each formed a distinct p85-associated complex. PY180 and PY52 each constituted about 10% of EGF-activated PI3-kinase, whereas PY105 was responsible for 80%. PY105 associated with Grb2 and SHP-2, and although it behaved like Gab1, none of the latter was detected in rat liver. We therefore cloned a cDNA from rat liver, which was found to be 95% homologous to the mouse Grb2-associated binder 2 (Gab2) cDNA sequence. Using a specific Gab2 antibody, we demonstrated its expression in and association with p85, SHP-2, and Grb2 upon EGF treatment of rat hepatocytes. Gab2 accounted for most if not all of the PY105 species, since immunoprecipitation of Gab2 with specific antibodies demonstrated parallel immunodepletion of Gab2 and PY105 from the residual supernatants. We also found that the PI3-kinase activity associated with Gab2 was totally abolished by dominant negative p85. Thus, Gab2 appears to be the principal EGF-induced PY protein recruiting and activating PI3-kinase and mitogenesis.     

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.     

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.     

Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts.

Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have developed a sensitive method to measure PI 3-kinase activity in diluted, detergent-containing whole cell extracts and used this method to determine total, soluble, and membrane-associated PI 3-kinase activity in PDGF-stimulated NIH 3T3 fibroblasts. PDGF stimulation induced a 1.4-fold increase in total Nonidet P-40-extractable PI 3-kinase activity, which occurred within 1 min and was maintained above basal levels at 10 min. At the same time, PI 3-kinase activity in the soluble fraction decreased 30-50%. However, membrane-bound PI 3-kinase activity increased 2.4-fold at 1 min and 3.1-fold at 5 min. Translocation of the p85 PI 3-kinase subunit to the membrane was maximal at 10 min. These results suggest that PDGF-mediated activation of PI 3-kinase in membrane fraction results from initial intrinsic enzymatic activation followed by translocation from the cytosol.     

Modulation of Src Homology 3 Proteins by the Proline-Rich Adaptor Protein Shb

In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.     

Stromal cell-derived factor-1alpha induces tube-like structure formation of endothelial cells through phosphoinositide 3-kinase

Stromal cell-derived factor-1alpha (SDF-1alpha) is a CXC chemokine, which induces tube formation of endothelial cells. Although SDF-1alpha transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1alpha-mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1alpha-mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (immortalized murine brain capillary endothelial) cells). SDF-1alpha stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and G(i) protein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1alpha activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted Deltap85-8 cells), which lacks association with the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1alpha failed to activate PI3-kinase and to stimulate tube formation. SDF-1alpha-induced tube formation was inhibited by an antibody against murine vascular endothelial cadherin. The antibody as well as LY294002 attenuated SDF-1alpha-mediated compact cell-cell contact, which proceeded to tube formation. Taken together, SDF-1alpha induces compact cell-cell contact through PI3-kinase, resulting in tube formation of endothelial cells.     

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: role of alphaGMR

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with alphaGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the alphaGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.     

Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.     

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.     

Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent manner. The affinity of the interaction of Ras-GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3-kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP-dependent interaction of PI 3-kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3-kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3-kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3-kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.     

Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells.

The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3-kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit-mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion.