Rate of CRL4(CRBN) substrate Ikaros and Aiolos degradation underlies differential activity of lenalidomide and pomalidomide in multiple myeloma cells by regulation of c-Myc and IRF4.

Recent discoveries suggest that the critical events leading to the anti-proliferative activity of the IMiD immunomodulatory agents lenalidomide and pomalidomide in multiple myeloma (MM) cells are initiated by Cereblon-dependent ubiquitination and proteasomal degradation of substrate proteins Ikaros (IKZF1) and Aiolos (IKZF3). By performing kinetic analyses, we found that the downregulation or proteasomal degradation of Ikaros and Aiolos led to specific and sequential downregulation of c-Myc followed by IRF4 and subsequent growth inhibition and apoptosis. Notably, to ensure growth inhibition and cell death, sustained downregulation of Ikaros and Aiolos, c-Myc or IRF4 expression was required. In addition, we found that the half-maximal rate, rather than the final extent of Ikaros and Aiolos degradation, correlated to the relative efficacy of growth inhibition by lenalidomide or pomalidomide. Finally, we observed that all four transcription factors were elevated in primary MM samples compared with normal plasma cells. Taken together, our results suggest a functional link between Ikaros and Aiolos, and the pathological dysregulation of c-Myc and IRF4, and provide a new mechanistic understanding of the relative efficacy of lenalidomide and pomalidomide based on the kinetics of substrate degradation and downregulation of their downstream targets.

Exploring the underlying structure of mental disorders: cross-diagnostic differences and similarities from a network perspective using both a top-down and a bottom-up approach.

BACKGROUND: It has been suggested that the structure of psychopathology is best described as a complex network of components that interact in dynamic ways. The goal of the present paper was to examine the concept of psychopathology from a network perspective, combining complementary top-down and bottom-up approaches using momentary assessment techniques. METHOD: A pooled Experience Sampling Method (ESM) dataset of three groups (individuals with a diagnosis of depression, psychotic disorder or no diagnosis) was used (pooled N = 599). The top-down approach explored the network structure of mental states across different diagnostic categories. For this purpose, networks of five momentary mental states ('cheerful', 'content', 'down', 'insecure' and 'suspicious') were compared between the three groups. The complementary bottom-up approach used principal component analysis to explore whether empirically derived network structures yield meaningful higher order clusters. RESULTS: Individuals with a clinical diagnosis had more strongly connected moment-to-moment network structures, especially the depressed group. This group also showed more interconnections specifically between positive and negative mental states than the psychotic group. In the bottom-up approach, all possible connections between mental states were clustered into seven main components that together captured the main characteristics of the network dynamics. CONCLUSIONS: Our combination of (i) comparing network structure of mental states across three diagnostically different groups and (ii) searching for trans-diagnostic network components across all pooled individuals showed that these two approaches yield different, complementary perspectives in the field of psychopathology. The network paradigm therefore may be useful to map transdiagnostic processes.

p120 catenin is a key effector of a Ras-PKCɛ oncogenic signaling axis.

Within the family of protein kinase C (PKC) molecules, the novel isoform PRKCE (PKCɛ) acts as a bona fide oncogene in in vitro and in vivo models of tumorigenesis. Previous studies have reported expression of PKCɛ in breast, prostate and lung tumors above that of normal adjacent tissue. Data from the cancer genome atlas suggest increased copy number of PRKCE in triple negative breast cancer (TNBC). We find that overexpression of PKCɛ in a non-tumorigenic breast epithelial cell line is sufficient to overcome contact inhibition and results in the formation of cellular foci. Correspondingly, inhibition of PKCɛ in a TNBC cell model results in growth defects in two-dimensional (2D) and three-dimensional (3D) culture conditions and orthotopic xenografts. Using stable isotope labeling of amino acids in a cell culture phosphoproteomic approach, we find that CTNND1/p120ctn phosphorylation at serine 268 (P-S268) occurs in a strictly PKCɛ-dependent manner, and that loss of PKCɛ signaling in TNBC cells leads to reversal of mesenchymal morphology and signaling. In a model of Ras activation, inhibition of PKCɛ is sufficient to block mesenchymal cell morphology. Finally, treatment with a PKCɛ ATP mimetic inhibitor, PF-5263555, recapitulates genetic loss of function experiments impairing p120ctn phosphorylation as well as compromising TNBC cell growth in vitro and in vivo. We demonstrate PKCɛ as a tractable therapeutic target for TNBC, where p120ctn phosphorylation may serve as a readout for monitoring patient response.

RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy.

RNA interference (RNAi) entails the potential for novel therapeutic strategies through the silencing of disease-causing genes in vivo. However, recent studies have raised an issue regarding applicable routes of administration for small interfering RNA (siRNA) molecules as therapeutics. In this study, we demonstrate that liposomally formulated siRNA molecules, the so-called siRNA-lipoplexes, but not naked siRNAs, are delivered to the tumor endothelial cells in vivo by microscopy. In addition, functional intracellular delivery of formulated siRNA targeting the tumor suppressor PTEN is shown in endothelial cells of the liver and tumor. Finally, the therapeutic potential of systemically administered siRNA(CD31)-lipoplexes is established by inhibition of tumor growth in two different xenograft mouse models. Our findings corroborate the applicability of this liposomal siRNA delivery technology for inducing RNAi to modulate gene expression levels in angiogenesis-dependent processes. In addition, our results advocate CD31 as a promising therapeutic target for antiangiogenic intervention. Therefore, our study provides a basis for the development of antiangiogenic cancer therapies based on RNAi.

A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium.

For the application of RNA interference (RNAi) in vivo the functional delivery of short interfering RNAs (siRNAs) is still the major obstacle. Therefore, delivery technologies need to be established for the systemic application of RNAi in vivo. Here we report uptake, biodistribution and in vivo efficacy of siRNA molecules formulated into siRNA-lipoplexes. The applied formulation is based on complex formation of positively charged liposomes, a mixture of cationic and fusogenic lipids complexed with the negatively charged siRNA. We determined by fluorescence microscopy the temporal and spatial distribution of fluorescently labeled siRNA-lipoplexes, the body clearance and endothelial cell type specific uptake after single intravenous injection. Furthermore, by using siRNA molecules for targeting endothelia-specifically expressed genes, such as CD31 and Tie2, we were able to demonstrate downregulation of the corresponding mRNA and protein in vivo. Taken together, we show the applicability of this non-viral delivery technology for inducing RNAi in the vasculature of mice after systemic application.

Identification of novel effectors of invasive cell growth downstream of phosphoinositide 3-kinase.

Conventional approaches to identifying cancer targets are complicated by the chromosomal instability of tumour cells, and typically result in a large number of differentially expressed candidate genes with uncertain disease relevance. Here we present a novel approach which aims to elucidate the molecular changes that are induced after loss of tumour suppressor function. Using gene silencing tools, we mimic the loss of tumour suppressor function to identify key regulators of tumour initiation and progression. Loss of function of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) correlates with increased invasive cell growth due to the resulting chronic activation of the PI 3-kinase (phosphoinositide 3-kinase) pathway. Induced activation of PI 3-kinase either by inhibiting PTEN expression or by using p110*, a constitutively active PI 3-kinase, increased signalling and the invasive growth potential of cells. Using this unbiased approach we have identified novel downstream effectors of PI 3-kinase/PTEN signalling that mediate the behaviour of cells with a hyperactive PI 3-kinase pathway. These molecules represent candidate targets for therapeutic intervention in patients with PTEN-deficient tumours.

The I kappa B kinase (IKK) complex is tripartite and contains IKK gamma but not IKAP as a regular component.

A critical step in the activation of NF-kappa B is the phosphorylation of I kappa Bs by the I kappa B kinase (IKK) complex. IKK alpha and IKK beta are the two catalytic subunits of the IKK complex and two additional molecules, IKK gamma/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-kappa B signaling. IKAP and IKK gamma belong to distinct cellular complexes. Quantitative association of IKK gamma was observed with IKK alpha and IKK beta. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKK gamma or IKAP blocked tumor necrosis factor alpha induction of an NF-kappa B-dependent reporter construct, but IKAP in addition affected several NF-kappa B-independent promoters. Whereas specific down-regulation of IKK gamma protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-kappa B activation, a similar reduction of IKAP protein levels had no effect on NF-kappa B signaling. Using solely IKK alpha, IKK beta, and IKK gamma, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKK gamma is a stoichiometric component of the IKK complex, obligatory for NF-kappa B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-kappa B activation.

Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions.

Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase.

[Continuous spinal anesthesia in very elderly patients with high anesthesia risk in traumatologic-orthopedic and general surgery interventions]. / Kontinuierliche Spinalanästhesie bei Patienten in hohem Lebensalter und mit hohem Anästhesierisiko bei traumatologisch-orthopädischen und allgemeinchirurgischen Eingriffen.

Continuous spinal anaesthesia (CSA) was carried out via a 28-gauge spinal catheter in 154 surgical patients at the Department of Anaesthesiology and Critical Care at Radeberg Asklepios-ASB Hospital between May 1992 and March 1999. The method was used preferably in patients aged over 70 (mean age 82.3 years) with high general risk during anaesthesia (ASA III-IV) who underwent orthopaedic or general surgery of the lower limb and hypogastrium. Remarkably, an anaesthetic level of between Th 8 and Th 10 was achieved with the low initial dose of 7.5 mg of 0.5% hyperbaric bupivacaine. Only minimal cardiovascular and respiratory side-effects were observed in comparison to single shot spinal and general anaesthesia. In the whole series, no anaesthesia-related complications were seen. Another benefit of CSA is the option of applying a second dose with longer duration of surgery to keep the optimal anaesthetic level. In addition, the method is suitable for postoperative analgesia over a period of 2 to 3 days.

Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation.

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.

Inhibition of mSOS-activity by binding of phosphatidylinositol 4,5-P2 to the mSOS pleckstrin homology domain.

There are several recently reported examples of inositol phospholipids binding to pleckstrin homology (PH) domains of proteins. The PH domain of SOS, a guanine nucleotide exchange factor for Ras, binds to phosphatidylinositol 4,5 bisphosphate (PtdIns4,5P2). We found that binding of PtdIns4,5P2 to 6-his-tagged recombinant mSOS in vitro inhibits the ability of SOS to catalyze the association of GTP on p21RAS. This inhibition was specific for PtdIns4,5P2: a number of other phosphatidylinositols and phosphatidylserine failed to inhibit Ras GTP-association. We confirmed that the specificity of binding of PtdIns's to recombinant GST-SOS-PH domain is the same as the specificity of PtdIns's for inhibition of SOS activity: namely, that only PtdIns4,5P2 binds significantly to the SOS-PH domain. In addition, the inhibition of Ras GTP-binding is not blocked by excess free inositols suggesting that SOS binds to PtdIns4,5P2 with higher affinity than it binds to free inositols. Addition of SOS-PH domain protein prevented the inhibition of SOS by PtdIns4,5P2 as did addition of the high affinity PtdIns4,5P2-binding drug neomycin. This confirmed that SOS inhibition is mediated by the SOS-PH domain binding to the inositol moiety of PtdIns4,5P2. Binding of Grb2 to SOS did not prevent the inhibition of SOS by PtdIns4,5P2 suggesting that there must be another mechanism for regulating this inhibition. These findings show that the phospholipid PtdIns4,5P2 can suppress the activity of an enzyme involved in signal transduction and suggest that this inhibitory effect must be relieved when SOS is activated.

Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence.

Insulin regulates the expression of multiple hepatic genes through a conserved insulin response sequence (IRS) (CAAAAC/TAA) by an as yet undetermined mechanism. Protein kinase B/Akt (PKB/Akt), a member of the PKA/PKC serine/threonine kinase family, functions downstream from phosphatidylinositol 3'-kinase (PI3K) in mediating effects of insulin on glucose transport and glycogen synthesis. We asked whether PKB/Akt mediates sequence-specific effects of insulin on hepatic gene expression using the model of the insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Insulin lowers IGFBP-1 mRNA levels, inhibits IGFBP-1 promoter activity, and activates PKB/Akt in HepG2 hepatoma cells through a PI3K-dependent, rapamycin-insensitive mechanism. Constitutively active PI3K and PKB/Akt are each sufficient to mediate effects of insulin on the IGFBP-1 promoter in a nonadditive fashion. Dominant negative K179 PKB/Akt disrupts the ability of insulin and PI3K to activate PKB/Akt and to inhibit promoter activity. The IGFBP-1 promoter contains two IRSs each of which is sufficient to mediate sequence-specific effects of insulin, PI3K, and PKB/Akt on promoter activity. Highly related IRSs from the phosphoenolpyruvate carboxykinase and apolipoprotein CIII genes also are effective in this setting. These results indicate that PKB/Akt functions downstream from PI3K in mediating sequence-specific effects of insulin on the expression of IGFBP-1 and perhaps multiple hepatic genes through a conserved IRS.

Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons.

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.

Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis.

The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.

Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.

A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.

Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown.

Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.

Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes.

Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitope-tagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.

Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.