p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.     

Radicicol Potentiates Neurotrophin-Mediated Neurite Outgrowth and Survival of Cultured Sensory Neurons from Chick Embryo

Radicicol, an antifungal antibiotic with markedly low toxicity, is a potent inhibitor of the Src family of protein tyrosine kinases and causes morphological reversion of v-src-transformed fibroblasts. Recently, this antibiotic was also found to inhibit Raf kinase. In the present study, we found that nanomolar concentrations of radicicol (10 ng/ml) enhanced the survival and neurite outgrowth of neurons from embryonic chick dorsal root ganglia (DRGs) and sympathetic ganglia. It potentiated the trophic effects of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 on the cultured DRG neurons. This concentration of radicicol did not alter the tyrosine phosphorylation of Trk receptors or the activity of mitogen-activated protein (MAP) kinases. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not inhibit radicicol, excluding the involvement of PI3-kinase in the radicicol-dependent trophic actions. These results suggest that radicicol mediates neuronal growth presumably via a mechanism not involving the activation of Trk receptors, MAP kinase, or PI3-kinase.     

Glial cell line-derived neurotrophic factor increases intracellular calcium concentration. Role of calcium/calmodulin in the activation of the phosphatidylinositol 3-kinase pathway

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.     

GM1-induced activation of phosphatidylinositol 3-kinase: involvement of Trk receptors

The ganglioside GM1 promotes neuronal growth, differentiation, survival, phenotypic expression, and function restoration, by apparently interacting with neurotrophic factors and/or their receptors. In brain, GM1 activates the Trk receptors for neurotrophins and the Raf/MEK/ERK cascade in situ and in vivo . We have expanded these studies and explored whether GM1 recruits the phosphatidylinositol 3 (PI3)-kinase pathway in brain also. Incubating striatal slices with GM1 increased the activity of PI3-kinase in phosphotyrosine immunoprecipitates in a time- and concentration-dependent manner, and the response was blocked by the PI3-kinase inhibitors wortmannin and LY294002. PI3-kinase activation following GM1 was rapid and short lasting with an EC₅₀ of 5 μmol/L. There was a temporally parallel activation of the downstream PI3-kinase target Akt, which was prevented by PI3-kinase inhibition. PI3-kinase activity was found increased in Trk and Gab1 immunoprecipitates, and co-immunoprecipitation studies demonstrated the association of Trk and Gab1 after GM1 treatment. Enhanced PI3-kinase activity associated with Trk or Gab1 immunoprecipitates was blocked by the Trk inhibitor K252a. GM1 did not appear to transactivate Trk and did not alter the efflux of neurotrophins in striatal slices. Our findings suggest that GM1 induces activation of PI3-kinase that is, in part, mediated through Trk and Gab1.     

Brain-derived neurotrophic factor induces excitotoxic sensitivity in cultured embryonic rat spinal motor neurons through activation of the phosphatidylinositol 3-kinase pathway

Neurotrophic factors (NTFs) can protect against or sensitize neurons to excitotoxicity. We studied the role played by various NTFs in the excitotoxic death of purified embryonic rat motor neurons. Motor neurons cultured in brain-derived neurotrophic factor, but not neurotrophin 3, glial-derived neurotrophic factor, or cardiotrophin 1, were sensitive to excitotoxic insult. BDNF also induces excitotoxic sensitivity (ES) in motor neurons when BDNF is combined with these other NTFs. The effect of BDNF depends on de novo protein and mRNA synthesis. Reagents that either activate or inhibit the 75-kDa NTF receptor p75NTR do not affect BDNF-induced ES. The low EC50 for BDNF-induced survival and ES suggests that TrkB mediates both of these biological activities. BDNF does not alter glutamate-evoked rises of intracellular Ca2+, suggesting BDNF acts downstream. Both wortmannin and LY294002, which specifically block the phosphatidylinositol 3-kinase (PI3K) intracellular signaling pathway in motor neurons, inhibit BDNF-induced ES. We confirm this finding using a herpes simplex virus (HSV) that expresses the dominant negative p85 subunit of PI3K. Infecting motor neurons with this HSV, but not a control HSV, blocks activation of the PI3K pathway and BDNF-induced ES. Through the activation of TrkB and the PI3K signaling pathway, BDNF renders developing motor neurons susceptible to glutamate receptor-mediated cell death.     

Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.     

Midkine Inhibits Caspase-Dependent Apoptosis via the Activation of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase in Cultured Neurons

Midkine (MK) is a new member of the heparin-binding neurotrophic factor family. MK plays important roles in development and carcinogenesis and has several important biological effects, including promotion of neurite extension and neuronal survival. However, the mechanism by which MK exerts its neurotrophic actions on neurons has not been elucidated to date. We have established an apoptosis induction system by serum deprivation in primary neuronal cultures isolated from mouse cerebral cortices. Neuronal apoptosis induced by serum deprivation was accompanied by the activation of caspase-3. MK, when added into the culture medium, inhibited the induction of apoptosis and activation of caspase-3 in a dose-dependent manner. Extracellular signal-regulated kinase (ERK) and Akt were not activated by serum deprivation, whereas ERK and Akt were rapidly activated by addition of MK. In addition, the trophic actions of MK of suppressing apoptosis and suppressing the activation of caspase-3 were abolished by concomitant treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and with wort-mannin or LY294002, specific inhibitors of phosphatidyl-inositol 3-kinase (PI 3-kinase). These PI 3-kinase inhibitors also inhibited the activation of ERK in response to MK, demonstrating a link between ERK and the caspase-3 pathway that is modulated by the PI 3-kinase activation. These results indicate that the ERK cascade plays a central role in MK-mediated neuronal survival via inhibition of caspase-3 activation.     

B-Raf Inhibits Programmed Cell Death Downstream of Cytochrome c Release from Mitochondria by Activating the MEK/Erk Pathway

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.     

Neurotrophins protect against cytosine arabinoside-induced apoptosis of immature rat cerebellar neurons

Neurotrophin-induced neuroprotection against apoptosis was investigated using immature cultured cerebellar granule cells (CGC) from newborn rat pups. Apoptotic cell death induced by treatment with cytosine arabinoside (AraC) was confirmed by DNA fragmentation and quantified by cell survival assays. AraC was most effective in inducing apoptosis when added to CGC on the day of culture preparation, while less or no effect was observed when added at 24 or 48h after plating, respectively. Pretreatment of CGC cultures for 24h with brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3), robustly protected against AraC neurotoxicity. K252a, an inhibitor of the tropomyosin-related kinase (Trk) tyrosine kinase receptor family which showed no toxicity by itself, blocked BDNF protection of AraC-induced apoptosis in a concentration-dependent manner. Neither protein kinase C activation nor inhibition mimicked or affected BDNF protection against AraC neurotoxicity. BDNF, but not NT-3, treatment of immature CGC caused a marked, but transient activation of Akt through phosphatidylinositol (PI) 3-kinase. The neuroprotective effects of BDNF were suppressed by pretreatment with LY 294002 (a PI 3-kinase inhibitor). BDNF neuroprotection was also preceded by activation of mitogen activated protein kinase (MAPK) and suppressed by two MAPK/ERK (MEK)-selective inhibitors, PD 98059 and U-0126. Moreover, inhibitors of PI 3-kinase and MEK potentiated AraC-induced neurotoxicity. These results show that neurotrophins protect against AraC-induced apoptosis, at least in part, through TrkB-mediated activation of the PI 3-kinase/Akt and MEK signaling pathways.     

Activation of Phosphatidylinositol 3-Kinase, but Not Extracellular-Regulated Kinases, Is Necessary to Mediate Brain-Derived Neurotrophic Factor-Induced Motoneuron Survival

Chick embryo spinal cord motoneurons develop a trophic response to some neurotrophins when they are maintained in culture in the presence of muscle extract. Thus, after 2 days in culture, brain-derived neurotrophic factor (BDNF) promotes motoneuron survival. In the present study we have analyzed the intracellular pathways that may be involved in the BDNF-induced motoneuron survival. We have observed that BDNF activated the extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and the phosphatidylinositol (PI) 3-kinase pathways. To examine the contribution of these pathways to the survival effect triggered by BDNF, we used PD 98059, a specific inhibitor of MAP kinase kinase, and LY 294002, a selective inhibitor of PI 3-kinase. PD 98059, at doses that significantly reduced the phosphorylation of ERKs, did not show any prominent effect on neuronal survival. However, LY 294002 at doses that inhibited the phosphorylation of Akt, a down-stream element of the PI 3-kinase, completely abolished the motoneuron survival effects of BDNF. Moreover, cell death triggered by LY 294002 treatment exhibited features similar to those observed after muscle extract deprivation. Our results suggest that the PI 3-kinase pathway plays an important role in the survival effect triggered by BDNF on motoneurons, whereas activation of the ERK MAP kinase pathway is not relevant.     

Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells

Angiopoietin-1 is a unique growth factor which induces Tie2 receptor autophosphorylation and interaction with signal transduction molecules, GRB2 and p85 subunit of PI 3-kinase, but no detectable mitogenic response. Here we show that PI 3-kinase-dependent activation of Akt and attachment to extracellular matrix are required for angiopoietin-1-mediated endothelial cell survival. Apoptosis of growth factor-deprived cells grown in monolayer was decreased by angiopoietin-1 and correlated with Akt activation. In contrast, angiopoietin-1, bFGF or VEGF failed to protect cells in suspension culture. Ceramide, an intermediate of several apoptotic pathways, interferes with growth factor-mediated Akt activation. Ceramide induced endothelial cell death and abolished angiopoietin-1-mediated activation of Akt and the effect on cell survival. In addition, we found that PI 3-kinase activity is necessary for migration of endothelial cells in response to Angiopoietin-1. A transient activation of MAPK/ERKs was also detected within 10 min after stimulation with angiopoietin-1. In contrast to VEGF-mediated biological effects, inhibition of MAPK/ERKs by PD98059 in endothelial cells did not affect angiopoietin-1 mediated survival or migration. These findings indicate significant differences in intracellular signaling between VEGF and angiopoietin-1 and that PI 3-kinase lipid products are key mediators of the biological effects of angiopoietin-1.     

Ethanol Induces Apoptosis in Cerebellar Granule Neurons by Inhibiting Insulin-Like Growth Factor 1 Signaling

Abstract: The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 m M K⁺ to one containing 5 m M K⁺. IGF-1 protected granule neurons from apoptosis in medium containing 5 m M K⁺. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K⁺, and failed to potentiate cell death in the presence of 5 m M K⁺. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15–20%) was observed at 1 m M and was half-maximal at 45 m M . The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

Neuronal survival induced by neurotrophins requires calmodulin

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.     

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.     

Dual control of muscle cell survival by distinct growth factor-regulated signaling pathways

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.     

Class II phosphoinositide 3-kinase is activated by insulin but not by contraction in skeletal muscle.

While the role of the class IA phosphoinositide 3-kinase (PI 3-kinase) in insulin signaling is well established, little is known about the role of the class II PI 3-kinases. We show that insulin stimulation of intact rat soleus and epitrochlearis muscles causes a 3- to 4-fold increase in the activity of the wortmannin-resistant alpha isoform of the class II PI 3-kinase (PI3K-C2alpha). This activation is rapid and parallels the insulin-induced activation of the class IA PI 3-kinase associated with IRS-1 in these muscles. However, while contraction activated p38 Map kinase, it did not stimulate the activity of the class II PI 3-kinase. Therefore, activation of class II PI 3-kinase is unlikely to provide a mechanism that explains the fact that exercise-induced activation of glucose uptake is not blocked by wortmannin. However, the results suggest that activation of class II PI 3-kinase is likely to play a role in insulin signaling pathways in skeletal muscle.