Specific PI 3-kinase Inhibitor Research Articles

FcgammaRI activation of phospholipase Cgamma1 and protein kinase C in dibutyryl cAMP-differentiated U937 cells is dependent solely on the tyrosine-kinase activated form of phosphatidylinositol-3-kinase.

The human high affinity receptor for immunoglobulin G, FcgammaRI, in dibutyryl cyclic AMP (dbcAMP)-differentiated U937 cells, is coupled to the activation of phospholipase C (PLC) and the conventional protein kinase C (PKC) isoforms, alpha, beta, and gamma. Here we demonstrate that aggregation of FcgammaRI activates the tyrosine-kinase regulated form of phosphatidylinositol-3-kinase (PI-3-kinase) and that an increase of phosphatidylinositol trisphosphate (PIP3) is essential for the activation and translocation of PLCgamma1 in these cells. In addition, activation of the PKC isoforms was ablated by specific inhibitors of PI3-kinase or by overexpression of a dominant negative p85 subunit of PI3-kinase. The findings reported here demonstrate that PLCgamma1 and PKC activation by FcgammaRI are downstream of PI3-kinase, and that in contrast to cytokine primed cells, only the tyrosine-kinase activated isoform of PI3-kinase is coupled to FcgammaRI in dbcAMP-differentiated cells.

Phosphatidylinositol 3-kinase translocation to the nucleus is an early event in the interleukin-1 signalling mechanism in human osteosarcoma Saos-2 cells.

Interleukin 1 (IL-1) is a proinflammatory cytokine which can elicit proliferative, differentiative, or metabolic responses. The molecular mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus, although extensively studied, have not been completely elucidated. We previously demonstrated that human osteosarcoma Saos-2 cells incubated with IL-1 presented a rapid and transient increase of phospholipase C activity exclusively at the nuclear level. Moreover, we presented evidence that not only the canonical inositol lipid signalling pathway was involved, but also the D3-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-kinase) were affected. The results of this study indicate that in Saos-2 cells PI 3-kinase is recruited and activated by IL-1 receptor I (IL-1RI) through binding of the SH2 domains to the consensus sequence on the C-terminal tail of the receptor, and that Tyr-479 is essential for PI 3-kinase activation. Moreover, IL-1 treatment triggers PI 3-kinase translocation to the nucleus; this event is rapid and transient in cells expressing high levels of IL-1RI (Saos-2/IL-1R) as well as in untransfected cells, although to a lesser extent. The data, based on immunochemical and immunocytochemical quantitative methods, indicate that PI 3-kinase translocation to the nucleus depends on PI 3-kinase activation. In fact, inactivation by two independent mechanisms, addition of specific PI 3-kinase inhibitors, or overexpression of a mutant form of IL-1RI, resulted in a substantial inhibition of PI 3-kinase translocation to the nucleus. These data suggest that PI 3-kinase recruitment by the activated receptor is a limiting step in PI 3-kinase activation and nuclear translocation. This early event in the IL-1 signalling mechanisms confirms that D3 inositides, as well as canonical inositides produced by nuclear phospholipase C isoforms, are involved in this pathway of activation of transcription factors.

Etk/Bmx, a PH-domain containing tyrosine kinase, protects prostate cancer cells from apoptosis induced by photodynamic therapy or thapsigargin.

Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability confirmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A specific inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an efficient effector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.

Involvement of the rapamycin-sensitive pathway in the insulin regulation of muscle protein synthesis in streptozotocin-diabetic rats

Insulin resistance in 3-day streptozotocin (STZ)-treated rats was manifested by the lack of antiproteolytic action of insulin as well as by a reduction of its stimulatory effect on protein synthesis (-60% compared with the control group) in epitrochlearis muscle incubated in vitro. In the present study, we have investigated the diabetes-associated alterations in the insulin signalling cascade, especially the phosphatidylinositol-3 kinase (PI-3 kinase)/p70 S6 kinase (p70(S6K)) pathway, in rat skeletal muscle. LY 294002, a specific inhibitor of PI-3 kinase, markedly decreased the basal rate of protein synthesis and completely prevented insulin-mediated stimulation of this process both in control and diabetic rats. Thus, PI-3 kinase is required for insulin-stimulated muscle protein synthesis in diabetic rats as in the controls. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), had no effect on the basal rate of protein synthesis in either of the experimental groups. In control rats, the stimulatory action of insulin on muscle protein synthesis was diminished by 36% in the presence of rapamycin, whereas in diabetic muscles this reduction amounted to 68%. The rapamycin-sensitive pathway makes a relatively greater contribution to the stimulatory effect of insulin on muscle protein synthesis in diabetic rats compared with the controls, due presumably to the preferential decrease in the rapamycin-insensitive component of protein synthesis. Neither basal nor insulin-stimulated p70(S6K) activity, a signalling element lying downstream of mTOR, were modified by STZ-diabetes.

P70 S6 kinase activation is not required for insulin-like growth factor-induced differentiation of rat, mouse, or human skeletal muscle cells.

Insulin-like growth factors (IGFs) are potent stimulators of muscle differentiation, and phosphatidylinositol 3-kinase (PI 3-kinase) is an essential second messenger in this process. Little is known about the downstream effectors of the IGF/PI 3-kinase myogenic cascade, and contradictory observations have been reported concerning the involvement of p70 S6 kinase. In an attempt to clarify the role of p70 S6 kinase in myogenesis, here we have studied the effect of rapamycin on rat, mouse, and human skeletal muscle cell differentiation. Both insulin and IGF-II activated p70 S6 kinase in rat L6E9 and mouse Sol8 myoblasts, which was markedly inhibited at 1 ng/ml rapamycin concentrations. Consistent with previous observations in a variety of cell lines, rapamycin exerted a potent inhibitory effect on L6E9 and Sol8 serum-induced myoblast proliferation. In contrast, even at high concentrations (20 ng/ml), rapamycin had no effect on IGF-II-induced proliferation or differentiation. Indeed, neither the morphological differentiation, as assessed by myotube formation, nor the expression of muscle-specific markers such as myogenin, myosin heavy chain, or GLUT4 (glucose transporter-4) glucose carriers was altered by rapamycin. Moreover, here we extended our studies on IGF-II-induced myogenesis to human myoblasts derived from skeletal muscle biopsies. We show that, as observed for rat and mouse muscle cells, human myoblasts can be induced to form multinucleated myotubes in the presence of exogenous IGF-II. Moreover, IGF-II-induced human myotube formation was totally blocked by LY294002, a specific PI 3-kinase inhibitor, but remained unaffected in the presence of rapamycin.

The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes quiescent but not proliferating MG-63 human osteosarcoma cells to radiation

Recent genetic and biochemical studies indicate that DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (dsb) repair and V(D)J recombination. Since the catalytic subunit of DNA-PK (DNA-PKcs) has high sequence homology with phosphatidylinositol 3-kinase (PI 3-kinase), we examined the effect of wortmannin, a specific inhibitor of PI 3-kinase, on the survival of human tumor cells after X-irradiation. The present study demonstrates that wortmannin at 20 μM is an effective radiosensitizer of quiescent (Q), but not proliferating (P) cells. In addition, the rejoining of DNA dsb is significantly inhibited in Q, but not in P cells. Finally, we found that Q cell extracts have approximately five-fold less DNA-PK activity than those of P cells. After a 2 h exposure to wortmannin, the DNA-PK activity of Q cell extracts was considerably lower than that of P cells. This can explain why wortmannin sensitizes Q, but not P cells to radiation.

Differentiated Phenotype of Smooth Muscle Cells Depends on Signaling Pathways through Insulin-like Growth Factors and Phosphatidylinositol 3-Kinase

Under conventional culture conditions, smooth muscle cells display their phenotypic modulation from a differentiated to a dedifferentiated state. Here, we established a primary culture system of smooth muscle cells maintaining a differentiated phenotype, as characterized by expression of smooth muscle-specific marker genes such as h-caldesmon and calponin, cell morphology, and ligand-induced contractility. Laminin retarded the progression of dedifferentiation of smooth muscle cells. Insulin-like growth factors (IGF-I and IGF-II) and insulin markedly prolonged the differentiated phenotype, with IGF-I being the more potent. In contrast, serum, epidermal growth factor, transforming growth factors, and platelet-derived growth factors potently induced dedifferentiation compared with angiotensin II, arginine-vasopressin, and basic fibroblast growth factor. Using the present culture system, we investigated signaling pathways regulating a phenotype of smooth muscle cells. In cultured cells, IGF-I specifically activated phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, protein kinase B, but not mitogen-activated protein kinases. Specific inhibitors of PI3-kinase (wortmannin and LY294002) induced dedifferentiation of smooth muscle cells even when they were cultured on laminin under IGF-I-stimulated conditions. The sole effect of laminin to retard the dedifferentiation was completely blocked by anti-IGF-I antibody, and laminin promoted the endogenous expression of IGF-I in cultured cells. The reduced promoter activity of the caldesmon gene induced by platelet-derived growth factor BB was overcome by the forced expression of the constitutive active form of PI3-kinase p110alpha catalytic subunit. These findings suggest that an IGF-I signaling pathway through PI3-kinase plays a critical role in maintaining a differentiated phenotype of smooth muscle cells.

Prosaposin prevents programmed cell death of rat cerebellar granule neurons in culture

Prosaposin, the precursor of sphingolipid activator proteins (saposin A–D), has been reported to be a neurotrophic factor in vitro and in vivo. Prosaposin mRNA is transiently expressed at a high level in developing cerebellum during the period of granule cell proliferation and maturation, suggesting its significance during development of cerebellum. Here we investigated the neuroprotective effect of prosaposin on cerebellar granule neurons, exposing primary cerebellar granule cells to low K + which induced programmed cell death. Prosaposin rescued mature cerebellar granule neurons in a bimodal manner. A similar neuroprotective effect was obtained using TX14(A), a 14-mer neurotrophic peptide derivative of prosaposin. An additive neuroprotective effect was observed between BDNF and TX14(A), but not between IGF-1 and TX14(A). Prosaposin rescued 60% of nifedipine sensitive cerebellar granule neurons as well as IGF-1, while BDNF did not. Furthermore, the neuroprotective action of prosaposin was inhibited by LY294002, a specific inhibitor of PI 3-kinase. These findings indicated that prosaposin had a trophic effect upon newborn cerebellar granule cells and that the neuroprotective action was similar to that of IGF-1 rather than BDNF. Prosaposin may play a role in cerebellar development during programmed cell death of cerebellar neurons.

Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes.

The present study investigated whether activation of phosphatidylinositol 3-kinase (PI3-kinase) is involved in the stimulation of hypertrophic growth of adult ventricular cardiomyocytes under alpha- or beta-adrenoceptor stimulation. Adult ventricular rat cardiomyocytes were used either directly after isolation (day 1 culture) or after cultivation for 6 days in presence of 20% fetal calf serum (day 7 culture). PI3-kinase activity was determined in extracts of cardiomyocytes after immunoprecipitation with an antibody against the p85 subunit of PI3-kinase. The influence of PI3-kinase inhibition on myocardial growth was determined using the specific PI3-kinase inhibitors wortmannin and LY294002. In day 1 cultures alpha-adrenoceptor stimulation, but not beta-adrenoceptor stimulation caused activation of PI3-kinase. In response to alpha-adrenoceptor stimulation but not beta-adrenoceptor stimulation an acceleration of protein synthesis (incorporation of 14C-phenylalanine) and an increase in the total masses of cellular protein and RNA was observed. In these cultures inhibition of PI3-kinase attenuated the acceleration of protein synthesis and the increase in cellular masses of protein or RNA in response to alpha-adrenoceptor stimulation. In day 7 cultures alpha- and beta-adrenoceptor stimulation caused activation of PI3-kinase and increased protein synthesis. In these cultures inhibition of PI3-kinase attenuated the growth response to alpha- and beta-adrenoceptor stimulation. PI3-kinase activation via protein kinase C-dependent or cAMP-dependent pathways is required for hypertrophic growth of adult cardiomyocytes.

Growth Hormone and Prolactin Stimulate Tyrosine Phosphorylation of Insulin Receptor Substrate-1, -2, and -3, Their Association with p85 Phosphatidylinositol 3-Kinase (PI3-kinase), and Concomitantly PI3-kinase Activation via JAK2 Kinase

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.

Insulin and Insulin-like Growth Factor-I Receptor Mediated Differentiation of 3T3-F442A Cells into Adipocytes: Effect of PI 3-Kinase Inhibition

The ability of insulin and insulin-like growth factors (IGF-I and IGF-II) to induce differentiation of 3T3-F442A cells into adipocytes was examined at various hormone concentrations. Both insulin and the IGFs promoted differentiation at concentrations compatible with binding to their cognate receptors, suggesting that both insulin and IGF-I receptors are capable of promoting this differentiation. Adipocyte conversion of 3T3-F442A cells was completely blocked in the presence of LY294002, a specific inhibitor of PI 3-kinase, indicating that PI 3-kinase activity plays a crucial role in the initial signalling events that trigger this differentiation process.

Wortmannin Inhibits Repair of DNA Double-Strand Breaks in Irradiated Normal Human Cells

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells.

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 approximately 6 microM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3'-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells.

Etk/Bmx is the newest member of Btk tyrosine kinase family that contains a pleckstrin homology domain, an src homology 3 domain, an src homology 2 domain, and a catalytic domain. Unlike other members of the Btk family kinases, which are mostly hemopoietic cell-specific, Etk/Bmx is preferentially expressed in epithelial and endothelial cells. We first identified this kinase in prostate cancer [Robinson, D., He, F., Pretlow, T. & Kung, H. J. (1996) Proc. Natl. Acad. Sci. USA 93, 5958-5962). Here we report that Etk is engaged in phosphatidylinositol 3-kinase (PI3-kinase) pathway and plays a pivotal role in interleukin 6 (IL-6) signaling in a prostate cancer cell line, LNCaP. Our evidence that PI3-kinase is involved in Etk activation includes: (i) Wortmannin, a specific inhibitor of PI3-kinase, abolished the activation of Etk by IL-6; (ii) a constitutively active p110 subunit of PI3-kinase was able to activate Etk in the absence of IL-6; and (iii) a dominant negative p85 subunit of PI3-kinase mutant blocked the activation of Etk by IL-6. Interestingly, IL-6 treatment of LNCaP induced a remarkable neuroendocrine-like differentiation phenotype, with neurite extension and enhanced expression of neuronal markers. This phenotype could be abrogated by the overexpression of a dominant-negative Etk, indicating Etk is required for this differentiation process.

Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes.

We have assessed five signal transduction pathways to determine the role each might play in the malignant transformation of mammary epithelium initiated by neu, heregulin/NDF, TGFalpha, v-Ha-ras and c-myc in transgenic mice. The study involves a molecular and pharmacologic assessment of Erk/MAP kinase, Jnk/SAP kinase, PI 3-kinase, protein kinase C, and the Src-related kinases Lck and Fyn. Our results indicate that oncogenes capable of transforming mammary gland epithelium activate and require specific signal transduction pathways. For example, mammary tumors initiated by neu, v-Ha-ras and c-myc have high levels of active Erk/MAP kinase and their anchorage independent growth is strongly inhibited by PD098059, an inhibitor of Mek/ MAP kinase kinase. By contrast, Erk/MAP kinase activity is weak in tumors initiated by TFGalpha and heregulin/NDF and the corresponding cell lines are not growth inhibited by PD098059. Similarly, PI 3-kinase is strongly activated in neu, TGFalpha and heregulin/NDF initiated tumor cell lines, but not in c-myc or v-Ha-ras initiated tumor cell lines. The anchorage independent growth of all these tumor cell lines are, however, inhibited by the specific PI 3-kinase inhibitor LY294001. Further illustrating this oncogene-based specificity, PP1, a specific inhibitor of the Src-like kinases, Lck and Fyn, blocks anchorage-independent cell growth only in the TGFalpha initiated mammary tumor cell line. Taken together with additional observations, we conclude that certain oncogenes reliably require the recruitment/activation of specific signal transduction pathways. Such specific relationships between the initiating oncogene and a required pathway may reflect a direct activating effect or the parallel activation of a pathway that is a necessary oncogenic collaborator for transformation in the mammary gland. The work points to a molecular basis for targeting therapy when an initiating oncogene can be implicated; for example, because of amplification, increased expression, genetic alteration, or heritable characteristics.

Wortmannin, an inhibitor of phosphatidyl-inositol 3-kinase, induces oocyte maturation through a MPF-MAPK-dependent pathway

Wortmannin has been shown to be a non-competitive and irreversible inhibitor of PI3 kinase. For this reason, it has attracted considerable interest and it has been used, as a selective inhibitor of the PI3 kinase, for the study of signal transduction pathways in different systems including Xenopus oocytes. We show here that wortmannin itself is able to induce meiotic maturation at doses slightly higher that those required for complete inhibition of PI3 kinase. This effect was shown to be independent of the ability to inhibit PI3K since another unrelated PI3K inhibitor, LY294002, was unable to induce oocyte maturation at inhibitory concentrations for PI3 kinase. The mechanism for wortmannin-induced maturation involves the activation of maturation promoting factor (MPF) and MAP kinase activities in a time course that preceded the appearance of germinal vesicle breakdown. Thus, the pathway activated by wortmannin directly or indirectly affects other protein or proteins, besides PI3 kinase, responsible for its activity. This new target is placed independently or downstream of the PI3 kinase inhibition and upstream of protein synthesis. Moreover, the inhibition of either MPF or cAMP phosphodiesterase blocks wortmannin-induced maturation. We conclude that wortmannin may be a valuable tool for the study of the pathway leading to mitotic maturation of oocytes, but cannot be used as a specific PI3 kinase inhibitor.

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.

Osmotic Shock Stimulates GLUT4 Translocation in 3T3L1 Adipocytes by a Novel Tyrosine Kinase Pathway

Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin- and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin- and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130(cas), and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125(FAK) and p130(cas). These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.

Involvement of phosphatidylinositol-3 kinase in prevention of low K +-induced apoptosis of cerebellar granule neurons

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K +, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K + on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K + induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K +, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

Tyrosine kinase, phosphatidylinositol 3-kinase, and protein kinase C regulate insulin-stimulated NaCl absorption in the thick ascending limb

We have previously shown a direct stimulatory effect of insulin on NaCl absorption in the medullary thick ascending limb of Henle's loop (mTAL). To further investigate the signal transduction involved, we determined whether tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase), and/or protein kinase C (PKC) regulate insulin-stimulated NaCl absorption in the mTAL by in vitro microperfusion methods. In control experiments, insulin increased transepithelial voltage (V(te)) and net lumen-to-bath Cl- flux (J(Cl)). Genistein and methyl 2,5-dihydroxycinnamate, two specific tyrosine kinase inhibitors, abolished the effects of insulin. Wortmannin, a specific PI3-kinase inhibitor, inhibited the action of insulin. The effects of insulin also were inhibited by staurosporin and calphostin C, which are dissimilar inhibitors of PKC. These results indicate that insulin stimulates NaCl absorption in the mTAL through tyrosine kinase, PI3-kinase, and PKC-mediated mechanisms. Moreover, because we have reported previously that insulin causes no detectable change in cytosolic free Ca2+ in the mTAL cells, the present results also suggest that insulin-induced PKC activation is not related to inositol 1,4,5-triphosphate (IP3) production.