A function for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in fibroblasts during mitogenesis: requirement for insulin- and lysophosphatidic acid-mediated signal transduction

We have previously shown that phosphatidylinositol 3-kinase alpha (PI 3-Kalpha) (p85alpha-p110alpha) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185-9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kbeta (p85alpha-p110beta) during mitogenesis. By using antibodies specific to p110beta we showed that PI 3-Kbeta is expressed in NIH 3T3 cells. PI 3-Kbeta and PI 3-Kalpha have common features: PI 3-Kbeta is tightly associated with a protein serine kinase that phosphorylates p85alpha, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kbeta was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kalpha was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110beta into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kbeta plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kalpha and PI 3-Kbeta, suggesting that Ras uses other effectors for DNA synthesis.     

Aggregation of the human high affinity immunoglobulin G receptor (FcgammaRI) activates both tyrosine kinase and G protein-coupled phosphoinositide 3-kinase isoforms

Phosphoinositide 3-kinases (PI3-kinases) play an important role in the generation of lipid second messengers and the transduction of a myriad of biological responses. Distinct isoforms have been shown to be exclusively activated either by tyrosine kinase-coupled or G protein-coupled receptors. We show here, however, that certain nonclassical receptors can couple to both tyrosine kinase- and G protein-dependent isoforms of PI3-kinase: thus, aggregation of FcgammaRI, the human high affinity IgG receptor, on monocytes unusually leads to activation of both of these types of PI3-kinase. After aggregation of FcgammaRI, phosphatidylinositol 3,4, 5-triphosphate (PIP3) levels rise rapidly in interferon gamma-primed cells, reaching a peak within 30 sec. Moreover, and in contrast to the situation observed after stimulation of these cells with either insulin or ATP, which exclusively activate the tyrosine kinase- and G protein-coupled forms of PI3-kinase, respectively, PIP3 levels remain elevated up to 15 min after receptor aggregation. We show here that although the initial peak results from transient activation of the p85-dependent p110 isoform of PI-3kinase, presumably through recruitment of tyrosine kinases by the gamma chain, the later sustained rise of PIP3 results from activation of the G protein betagamma subunit-sensitive isoform, p110gamma. This finding indicates that receptors lacking an intrinsic signaling motif, such as FcgammaRI, can recruit both tyrosine kinase and G protein-coupled intracellular signaling molecules and thereby initiate cellular responses.     

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.     

The treatment of disseminated malignant melanoma with special reference to the role of interferons, vinca alkaloids and tamoxifen

Treatment of quiescent NIH3T3 cells with PDGF BB results in the transient activation and hyperphosphorylation of the protein-tyrosine kinase, c-Src. These effects correlate with novel serine and tyrosine phosphorylations in the N-terminal non-catalytic region of the molecule, which contains an SH3 and SH2 domain. In this study, a site of PDGF-induced tyrosine phosphorylation was mapped to Tyr 138 in the SH3 domain; Tyr 138 is exposed on the SH3 peptide binding surface. This same site is phosphorylated in vitro by the PDGF receptor when purified baculovirus-expressed c-Src is complexed with the activated receptor. Phosphorylation of Tyr 138 required association of c-Src with the PDGF receptor via its SH2 domain. When a c-Src Phe 138 mutant was stably expressed in Src- mouse fibroblasts, it was activated to the same extent as wild type c-Src following PDGF stimulation, indicating that phosphorylation of this site is not required for PDGF-mediated activation. However, Tyr 138 phosphorylation was found to diminish SH3 domain peptide ligand binding ability in vitro.     

SHP-1 associates with both platelet-derived growth factor receptor and the p85 subunit of phosphatidylinositol 3-kinase

The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.     

Lovastatin disrupts early events in insulin signaling: a potential mechanism of lovastatin's anti-mitogenic activity

The mechanism by which lovastatin lowers cholesterol levels is well characterized but little is known about its anti-mitogenic and anti-tumorigenic mechanism. Here we demonstrate that lovastatin disrupts early events in the mitogenic signaling pathways of insulin. Insulin treatment (200 mM) of quiescent HIR rat-1 fibroblasts results in an 8-fold stimulation of phosphatidylinositol-3-kinase (PI-3-K) activity. Overnight pretreatment of cells with lovastatin (20 microM) inhibits insulin stimulation of PI-3-K activity by 75%. Immunoprecipitation and immunoblotting experiments using antibodies against the regulatory subunit of PI-3-K (p85), phosphotyrosine, and insulin receptor alpha and beta subunits demonstrate that lovastatin inhibits the association of p85 with tyrosine phosphorylated insulin receptor substrate-1 and the beta subunit of the insulin receptor. Furthermore, lovastatin dramatically reduces (70-100%) the level of tyrosine phosphorylated insulin receptor beta subunit following insulin stimulation. These results clearly demonstrate that lovastatin disrupts early events of insulin mitogenic signaling by reducing the levels of tyrosine phosphorylated beta subunit and suggest that this disruption is a potential mechanism for the anti-mitogenic effect of lovastatin.     

Mutagen sensitivity to benzo(a)pyrene diol epoxide and the risk of squamous cell carcinoma of the head and neck

Phosphatidylinositol (PI)-3 kinase is an enzyme that phosphorylates D3 position of PI and its derivatives. It is activated immediately after growth factor or differentiation factor stimulation, suggesting that PI-3 kinase is involved in signal transduction of the stimulations. PI-3 kinase appears to play important roles in various points including signaling to the nucleus, vesicle transport, and rearrangement of cytoskeleton since many cell responses which requires these events were affected by inhibition or activation of PI-3 kinase. There are many mechanisms of activation of PI-3 kinase including binding to phosphotyrosine containing proteins or binding to Ras. Each mechanism can target PI-3 kinase to the right place to induce the right cell responses.     

Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110beta is synergistically activated by the betagamma subunits of G proteins and phosphotyrosyl peptide

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the betagamma subunits of G protein (Gbetagamma) from rat liver. The Gbetagamma-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110beta and p85 (or their related subunits). The stimulation by Gbetagamma was inhibited by the GDP-bound alpha subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gbetagamma was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110beta/p85alpha expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110alpha and p85 in the same rat liver, together with a recombinant p110alpha/p85alpha, was not activated by Gbetagamma, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110beta/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.     

Requirement for both Shc and phosphatidylinositol 3' kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis

Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3' (PI-3') kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3' kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3' kinase were highly apoptotic, suggesting that recruitment of PI-3' kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3' kinase binding site was noted in tumors derived from the strains expressing the PI-3' kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3' kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3' kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.     

Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors

In order to study the regulation of the ribosomal protein S6 kinase, p70s6k, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha2C10 receptor, with the alpha2 agonist UK 14304 led to a transient increase in p70s6k activity. UK 14304 also activated p70s6k in a clone expressing the porcine alpha2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70s6k in alpha2 receptor-transfected and in nontransfected cells. Activation of p70s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70s6k by both agents. Activation of p70s6k by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70s6k by UK 14304, LPA, and PDGF. These data indicate that p70s6k is regulatable by Gi-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70s6k by such agents appears to involve an isoform of PI 3-kinase.     

Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.     

Signal transduction by the PDGF receptors

The three isoforms of PDGF bind with different affinities to two related tyrosine kinase receptors, denoted the PDGF alpha- and beta-receptors. Ligand binding induces receptor dimerization, creating receptor homo- or heterodimers. Dimerization is accompanied by, and might be a prerequisite for, receptor autophosphorylation and kinase activation. Receptor autophosphorylation serves to regulate the kinase activity and to create binding sites on the receptor molecule for downstream signalling components. The activities of the signalling components are ultimately manifested as specific biological responses. All the currently described PDGF receptor-binding components, e.g. phospholipase C-gamma, members of the src family of cytoplasmic tyrosine kinases, the rasGT-Pase activating protein and p85, the regulatory subunit of phosphatidylinositol 3' kinase, contain a conserved src homology 2-domain, through which the association with the receptor takes place. The receptor-binding components appear to either possess an intrinsic enzymatic activity, or they function as adaptors, which may complex with catalytically active components. For most receptor-binding components, there is insufficient understanding of how binding to the receptor affects the catalytic function. Certain of these components become tyrosine-phosphorylated, i.e. they are substrates for the receptor tyrosine kinase. Moreover, the change in subcellular localization, which most of the receptor binding components undergo in conjunction with receptor binding, could play a critical role. The current efforts of many laboratories are aimed at delineating different PDGF receptor signal transduction pathways and what roles the different receptor-binding components play in the establishment of these pathways.     

Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase

We have previously demonstrated that interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely related or identical to the c-fes proto-oncogene product (FES) and association of this protein with the IL-4 receptor alpha chain (IL-4R alpha). IL-4 is known to induce association of phosphatidylinositol-3 (PI3) kinase with the IL-4R alpha. Since FES contains the consensus motifs for PI3 kinase binding, we tested the possibility that FES may associate with PI3 kinase upon IL-4 stimulation. We demonstrate herein that IL-4 stimulation induced rapid association of FES or a related protein with PI3 kinase in mouse T-cell lines. We also show an association of human FES (hFES) with the src homology 2 (SH2) domain of PI3 kinase in a COS7 cell expression system. The in vitro PI3 kinase assay using COS7 cells suggested that hFES partly contributes to the association between the hIL-4R alpha and PI3 kinase. We have further identified the important region in the cytoplasmic domain of the hIL-4R alpha for association of tyrosine-phosphorylated hFES with the hIL-4R alpha and SH2 domain of PI3 kinase using a COS7 cell expression system. These results suggest that FES or a related protein/PI3 kinase pathway may play a role in the pleiotropic effects of IL-4.     

Heat shock activates c-Src tyrosine kinases and phosphatidylinositol 3-kinase in NIH3T3 fibroblasts

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.     

IL-2, but not IL-4 and other cytokines, induces phosphorylation of a 98-kDa protein associated with SHP-2, phosphatidylinositol 3'-kinase, and Grb2

Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.     

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2

Recently c-Cbl has been reported to be phosphorylated upon CSF-1 stimulation. The product of the c-cbl proto-oncogene (c-Cbl) is a 120 kDa protein harboring several docking sites for Src homology 2 (SH2) domain containing proteins and proline-rich regions that have been shown to allow its constitutive association with the SH3 domains of Grb2. We demonstrate here that CSF-1 exposure of stable transfectant CHO cells expressing the CSF-1 receptor induced the sustained tyrosine phosphorylation of c-Cbl and its subsequent association with Crk-II and the p85 kDa subunit of the PI 3-kinase, while it constitutively associates with Grb2. We demonstrate by in vitro experiments that these associations require the SH2 domain of Crk-II and both the C- and N-terminal SH2 domains of the p85 subunit of the PI 3-kinase. cCbl is the major PI 3-kinase-containing protein in c-Fms expressing CHO cells upon CSF-1 stimulation. Thus c-Cbl behaves as a core protein, allowing the formation of a quaternary complex including, Crk-II, PI 3-kinase and Grb2. We provide evidence that this multiprotein complex can interact with the tyrosine phosphorylated CSF-1 receptor through the unoccupied SH2 domain of Grb2.     

p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells

Flt3/Flk2 belongs to class III receptor tyrosine kinases. Like other members of type III receptor tyrosine kinases, murine Flt3 induces tyrosine phosphorylation of p85 and subsequently activation of PI3 kinase upon FL binding. While p85 binds murine Flt3 at Y958 in the carboxyl terminus of the receptor, human Flt3 does not have a potential p85-binding site in the carboxyl terminus. In this study, we examined whether p85 binds to human Flt3 in Baf3/Flt3 and THP-1 cells. In contrast to murine Flt3, p85 is not tyrosine phosphorylated after FL stimulation, nor does it bind Flt3 in both cell lines. Instead p85 associates inducibly with tyrosine phosphorylated SHP-2 and constitutively with SHIP and two tyrosine phosphorylated proteins with molecular weights about 100-kDa (p100) and 120-kDa (p120) in Baf3/Flt3 cells. The p100 associates with both p85 and SHP-2. In THP-1 cells, p85 associates inducibly with tyrosine phosphorylated SHIP, p100 and p120. These results indicate that p85 does not bind human Flt3, but forms a complex with SHP-2, SHIP, p100 and p120 in hematopoietic cells.     

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways

We have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C2C12 myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C2C12 myotubes with insulin (100 nmol/l, 5 min) or leptin (0.62 nmol/l, 10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulin-receptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C2C12 myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C2C12 myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform.     

Mitochondrial malic enzyme: purification from bovine brain, generation of an antiserum, and immunocytochemical localization in neurons of rat brain

The receptor kinase activity associated with the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor plays an important role in ligand-induced signaling events. The effect of specific, synthetic chemical inhibitors of PDGF- and EGF-mediated receptor tyrosine autophosphorylation on receptor signaling were examined in NIH 3T3 cells overexpressing PDGF or EGF receptors. Specific inhibition of ligand-dependent receptor autophosphorylation, PI3K activation, mitogen-activated protein kinase (MAPK) activation, cyclin E-associated kinase activity and cell proliferation was measured after treatment of cells with these inhibitors. A synthetic PDGF receptor kinase inhibitor exhibited specific inhibitory properties when tested for PDGF-induced receptor autophosphorylation, MAPK activity, PI3K activation, entry into S phase and cyclin E-associated kinase activity. A synthetic EGF receptor kinase inhibitor showed selective inhibitor properties when tested for EGF-induced receptor autophosphorylation, MAPK activation, PI3K activation, entry into S phase and cyclin E-associated kinase activity. In both cases, these compounds were found to be effective as inducers of growth arrest and accumulation of cells in the G1 phase of the cell cycle after ligand treatment. However, at high concentrations, the EGF receptor kinase inhibitor was observed to exhibit some nonspecific effects as demonstrated by attenuation of PDGF-induced receptor autophosphorylation and cell cycle progression. This demonstrates that it is critical to use the lowest concentration of such an inhibitor that will alter the response under investigation, to have confidence that the conclusions derived from the use of such inhibitor are valid. We conclude that these experimental parameters signify useful end points to measure the relative selectivity of tyrosine kinase inhibitors that affect receptor-mediated signal transduction.