Article |
Address correspondence to Joan X. Comella, Grup de Neurobiologia Molecular, Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Avda Rovira Roure, 44, 25198 Lleida, Spain. Tel.: 34-973-702414. Fax: 34-973-702438. E-mail: joan.comella{at}cmb.udl.es
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Abstract |
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Key Words: calmodulin; neurotrophin; cell survival; PKB; motoneuron
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Introduction |
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The pathway that leads to the activation of the PI 3-kinase after Trk activation begins with the autophosphorylation of the receptor at the residue Tyr490 and the phosphorylation of Shc (Obermeier et al., 1993; Hallberg et al., 1998). This allows the recruitment to the membrane of several adaptor proteins, which upon tyrosine phosphorylation interact with and activate PI 3-kinase (Holgado-Madruga et al., 1997; Yamada et al., 1997). Furthermore, it has been reported that Ras cooperates in the activation of the PI 3-kinase, since dominant negative forms of this GTPase inhibit PI 3-kinase activity (Rodriguez-Viciana et al., 1994). PI 3-kinase catalyzes the phosphorylation of the D3 hydroxyl group of the inositol ring of phosphoinositides (PtdIns) (Czech, 2000). PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are the main products generated and provide docking sites for pleckstrin homology (PH) domains of effector proteins such as the Ser/Thr protein kinase B (PKB), the cellular homologue of the transforming oncogene v-Akt (Bellacosa et al., 1991; Coffer and Woodgett, 1991; Jones et al., 1991). The interaction of PtdIns-3,4-P2/PtdIns-3,4,5-P3 with PKB allows the translocation of the protein to the plasma membrane where it becomes fully activated upon phosphorylation at two residues, Thr308 and Ser473 (Alessi et al., 1996).
In a variety of cell systems, including neuronal cells, PKB mediates an important part of the trophic signal derived from PI 3-kinase activation (Dudek et al., 1997; Philpott et al., 1997; Crowder and Freeman, 1998). Several studies have reported that PKB interferes with the cell death machinery phosphorylating and inactivating proteins that are directly involved in the induction of apoptosis such as GSK3ß, BAD (a member of the Bcl-2 family of proteins), or members of the Forkhead family of transcription factors involved in the transcription of Fas ligand (Datta et al., 1999).
Bioelectrical activity cooperates with NTs in promoting neuronal survival during development (Franklin and Johnson, 1992). Neuronal activity exerts its trophic effects by moderately increasing the intracellular Ca2+ concentration ([Ca2+]i). Ca2+ triggers the activation of similar signaling pathways to those activated by NTs, mainly through the Ca2+ receptor protein calmodulin (CaM) (Finkbeiner and Greenberg, 1996). Moreover, it has been reported that activation of Trk leads to a small and rapid increase of [Ca2+]i (Pandiella-Alonso et al., 1986; Jiang and Guroff, 1997). However, the involvement of Ca2+ in the response of the cells to the NTs has been poorly characterized. In the present work, we show that CaM is necessary for the promotion of cell survival triggered by NTs in PC12 cells and in chicken spinal cord motoneurons (MTNs). Our results demonstrate that this effect is mainly due to the regulation of PKB activity. We provide evidence that CaM is necessary to detect PtdIns-3,4-P2/PtdIns-3,4,5-P3 in the plasma membrane of live cells thus providing a possible mechanism by which CaM regulates PKB activity and cell survival.
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Results |
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CaM antagonists block NT-induced cell survival
The survival induced by NGF in PC12 cells can be strongly attenuated by inhibitors of PI 3-kinase (Yao and Cooper, 1995) or by treatments that block PKB activity (Weihl et al., 1999; Salinas et al., 2000). Therefore, we wanted to analyze whether the inhibition of PKB induced by CaM antagonists had any effect on cell survival. For this purpose, we maintained PC12 cell cultures in the presence of NGF (10 ng/ml) plus W13 (30 mM) or LY294002 (20 mM) for 15 h. At the end of the treatments, cells were fixed and stained with Hoechst 33258 or subjected to a TdT-mediated dUTP nick end labeling (TUNEL) assay. As shown in Fig. 2 A, the percentage of cells that display typical morphological apoptotic nuclei in the cultures treated with NGF plus W13 increased significantly when compared with cultures treated with NGF alone. Values were similar to those obtained in cultures maintained without any trophic support or in cultures treated with NGF plus LY294002. The values of apoptotic nuclei in W13 treatments were indistinguishable from those observed in starved cultures or in cultures treated with NGF plus LY294002 (Fig. 2 B). Similarly, W13 increased the number of TUNEL-positive cells in cultures maintained with NGF (5.98 ± 1.04%) when compared with cells treated with NGF alone (3.26 ± 0.37%), reaching similar values to those observed in cultures treated with NGF plus LY294002 (7.01 ± 0.84%). (In these experiments, the percentage of TUNEL-positive cells in cultures without trophic support was 9.96 ± 1.17) [Fig. 2 C].) The concentration of W13 used in these experiments did not increase the cell death observed in trophic support-starved cultures, suggesting that W13 was specifically interfering with the intracellular signaling pathway involved in NGF-induced cell survival (Fig. 2 A). As a control, we checked whether the concentration of W13 used in the experiments of survival (30 µM) inhibited the activation of PKB induced by 10 ng/ml NGF. As shown in Fig. 2 D, this concentration of the drug induced an effective and sustained inhibition of both Thr308 and Ser473 phosphorylations of PKB.
We next analyzed the involvement of CaM in cell survival in a primary culture of neurons, and for this we used chicken MTNs maintained with BDNF. This experimental paradigm has several similarities with PC12 cells maintained with NGF. For example, we have demonstrated previously that BDNF-induced MTN survival is prevented by LY294002 (Dolcet et al., 1999), and here we have shown that the activation of PKB triggered by BDNF requires Ca2+ and CaM (Fig. 1 F). Therefore, MTNs were treated with BDNF (10 ng/ml) plus W13 (30 µM), and cell survival was analyzed after 24 h. Our experiments showed that W13 but not W12 prevented the BDNF-induced MTN survival without increasing the cell death of parallel control cultures maintained in basal media without BDNF (Fig. 3 A). This effect correlated with a significative increase in the number of TUNEL-positive cells (Fig. 3, B and D) and in the number of the cells displaying typical morphological apoptotic nuclei (Fig. 3 B). As shown in Fig. 3 C, the morphology of the apoptotic nuclei in W13 treatments was indistinguishable from that observed in starved cultures or in cultures treated with BDNF plus LY294002. Fig. 3 E shows that 30 µM of W13 effectively blocked the phosphorylation of PKB induced by 10 ng/ml BDNF.
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Discussion |
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We have observed that extracellular Ca2+ blockade did not affect the activation of PKB induced by NGF in PC12 cells thus confirming previous observations obtained in Balb/c-3T3 fibroblasts stimulated with EGF (Conus et al., 1998). In contrast, our results point out that intracellular Ca2+ is completely necessary for the activation of PKB. This effect seems to be mediated by CaM, since CaM inhibition mimicked the effects of intracellular Ca2+ blockade on PKB activation. It has been reported previously that Trk activation induces a small and rapid increase of [Ca2+]i (Jiang and Guroff, 1997). This is probably achieved through the activation of PLC and the subsequent release of Ca2+ from intracellular stores through inositol 1,3,4-triphosphate receptors (Obermeier et al., 1996). This mechanism seems to participate in the activation of the ERK MAP kinases, since TrkA receptors that carry a point mutation in the tyrosine that binds PLC
(TrkAY785F) fail to completely activate these kinases (Stephens et al., 1994). However, it is less clear whether this IP3-dependent Ca2+ release could participate in the activation of PKB induced by Trk. For instance, NGF effectively activates PKB in Rat-1 cells expressing TrkAY785F (Ulrich et al., 1998). Furthermore, the inhibition of IP3 receptors with the specific inhibitor 2-APB (Maruyama et al., 1997) does not block the activation of PKB induced by NGF in PC12 cells (unpublished data). On the basis of these observations, it is possible that an IP3-independent mechanism of intracellular Ca2+ release induced by Trk could be involved in the activation of PKB. Alternatively, it could be also possible that basal levels of [Ca2+]i are required to activate PKB.
It has been reported that membrane depolarization promotes cell survival in rat sympathetic neurons and granule neurons through a mechanism involving CaMKII and PI 3-kinase (Hack et al., 1993; Vaillant et al., 1999; Ikegami and Koike, 2000). However, we have not observed any effect of KN-62, a specific inhibitor of CaMKII, on the activation of PKB induced by NGF in PC12 cells (unpublished data). According to this result, it has been reported that KN-62 does not block cell survival of sympathetic neurons maintained with NGF (Vaillant et al., 1999; Ikegami and Koike, 2000). Results obtained in depolarized cerebellar granule neurons have provided contradictory conclusions regarding the participation of the PI 3-kinase in the depolarization-induced cell survival (D'Mello et al., 1997; Dudek et al., 1997; Miller et al., 1997). However, it seems clear that depolarization-induced survival is a CaM-dependent PI 3-kinase-independent phenomenon in chicken MTNs (Soler et al., 1998). These CaM-dependent PI 3-kinase-independent mechanisms have been attributed recently to the CaMKK. It has been reported that N-methyl-D-aspartateinduced increase of [Ca2+]i in NG108 neuroblastoma cells activates CaMKK, which in turns directly phosphorylates and activates PKB (Yano et al., 1998). In this study, we have analyzed the participation of CaMKK in PKB activation, and we have observed that CaMKKK157A, a dominant negative form of the enzyme, did not abolish the activation of PKB induced by NGF, even at low nonsaturant concentrations of the NT (that is, 5 ng/ml). Moreover, CaMKKK157A did not have any effect on NGF-induced PC12 cell survival (unpublished data). According to these results, the promotion of cell survival induced by insulin-like growth factor in NG108 neuroblastoma cells was not affected by CaMKKK157A (Yano et al., 1998). Together, these results suggest that the CaM-dependent mechanisms involved in the promotion of cell survival induced by increases of [Ca2+]i (for example, due to membrane depolarization or Ca2+-mobilizing agonists) are distinct from those involved in cell survival promoted by NTs.
To ascertain the mechanism(s) whereby CaM modulates PKB activity in NT-treated cells, we explored some of the upstream steps involved in its activation. Neither the phosphorylation of Trk or Shc, the interaction of Shc with Grb2, nor the activation of Ras induced by NGF in PC12 cells required CaM (Egea et al., 2000). In the present work, we have also analyzed the activation of PI 3-kinase in antiP-Tyr immunoprecipitates in NGF-stimulated PC12 cells and in BDNF-stimulated MTNs, concluding that it was not affected specifically by the treatment with CaM antagonists. However, as judged by the lack of peripheral distribution of the EGFPPKBPH fusion protein CaM antagonists did not allow to detect PtdIns-3,4-P2/PtdIns-3,4,5-P3 in the plasma membrane of intact cells after NGF and BDNF stimulation. These apparently contradictory results obtained using in vitro and in vivo assays have also been observed in 3T3L1 adipocytes. In these cells, CaM antagonists prevent the presence of PtdIns-3,4-P2/PtdIns-3,4,5-P3 in the plasma membrane induced by insulin or PDGF without affecting the PI 3-kinase activity measured in antiP-Tyr immunoprecipitates (Yang et al., 2000). Therefore, these results seem to indicate that CaM is necessary for the generation and/or stabilization of the PI 3-kinase products. Several mechanisms can account for the role of CaM in this effect. For example, it can be possible that CaM exerts a regulation of PI 3-kinase activity in intact cells that is undetectable in the in vitro kinase assays. Such an explanation has been reported in the regulation of the PI 3-kinase by Ras. Dominant negative forms of this GTPase interact with and inhibit the activity of the PI 3-kinase, but this is only detectable in in vivo assays. In antiP-Tyr immunoprecipitates, the interaction is lost, and therefore the inhibitory effect of the dominant negative forms of Ras cannot be observed (Rodriguez-Viciana et al., 1994). Another explanation could be that CaM can be required for the localization of the PI 3-kinase close to its substrates in intact cells. Indeed, CaM has been involved in the appropriate intracellular targeting of several proteins such as Rad or p21(Cip1) (Moyers et al., 1997; Taules et al., 1999). These hypothesis are supported by recent findings, showing that both the p85 and p110 subunits of the PI 3-kinase are able to interact with CaM in a Ca2+-dependent manner (Joyal et al., 1997; Fischer et al., 1998). Finally, the possibility that CaM could negatively regulate the activity of the phosphatase(s) involved in the downregulation of the products generated by the PI 3-kinase cannot be ruled out. In this case, the inhibition of CaM would accelerate the disappearance of PtdIns-3,4-P2/PtdIns-3,4,5-P3 from the plasma membrane.
Our findings have relevant physiological implications in the developing nervous system. It is well known that increases of [Ca2+]i underlying neuronal activity cooperate with NTs to promote neuronal survival. This phenomenon ensures that the correct and functional connections will be selected over the aberrant and nonfunctional ones (Franklin and Johnson, 1992). This phenomenon can be reproduced in vitro. For instance, levels of membrane depolarization that by themselves do not have any trophic effect synergize with limited amounts of NGF to promote cell survival in cultures of sympathetic neurons (Vaillant et al., 1999). Our results suggest a mechanism that could explain the synergistic effect of membrane depolarization on NT-induced cell survival in which Ca2+ and CaM would sensitize the pathway involved in the activation of PKB and in the promotion of cell survival. Moreover, since Ca2+ and CaM are also necessary for the activation of the ERK/MAP kinases induced by NTs (Egea et al., 2000) we propose that Ca2+ and CaM play a central role in the regulation of the intracellular signaling pathways activated by NTs.
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Materials and methods |
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Total cell lysates were obtained solubilizing the cells in 2% SDS and 125 mM Tris, pH 6.8, and sonicated. For immunoprecipitations, kinase assays, or Ras activity, cells were lysed at 4°C in the adequate lysis buffer (see below), and nuclei and cellular debris were removed by microfuge centrifugation. Protein concentration in cell lysates was quantified using the Bio-Rad Laboratories Dc protein assay.
Plasmids and cell transfection
CaMKK and PKB cDNAs were cloned from PC12 total RNA using the RobusT reverse transcription-PCR kit (Finnzymes). The dominant negative form (CaMKKK157A) and the constitutive active form (CaMKK1413) of CaMKK have been described previously (Enslen et al., 1996; Yano et al., 1998). CaMKKK157A point mutant was generated using the QuickChange Site-directed mutagenesis kit (Stratagene) and sequenced to confirm the mutation. CaMKK1413 was generated by PCR, amplifying the sequence that codifies for the first 413 amino acids of the protein using the DyNAzyme EXT DNA polymerase (Finnzymes). NH2-terminally tagged green fluorescent protein EGFPPKBPH was constructed incorporating a fragment of 750 bp, encoding the first 250 amino acids of PKB
(containing the PH domain) with the EGFP as described previously (Currie et al., 1999). All of the constructs were subcloned into the mammalian expression vector pcDNA3 (Invitrogen). DNA purification for cell transfection was performed using the QIAGEN Plasmid Maxi kit.
PC12 cells were transfected by electroporation as described in Espinet et al. (2000). MTNs were transfected 3 h after purification using the Lipofectamine 2000 reagent as suggested by the manufacturer (Life Technologies). When indicated, PC12 cells or MTNs were cotransfected with pEGFP (CLONTECH) and pSG5-Gag-PKB, pCMV5-HA-PKBT308D/S473D, or the empty vector, using a one to four molar ratio.
Evaluation of cell survival
PC12 cells cultured in serum-containing medium were changed to a serum-free medium containing NGF (10 ng/ml) plus the indicated inhibitors. After 15 h, cells were fixed and stained with the DNA dye Hoechst 33258 as described previously (Dolcet et al., 1999). When indicated, cells were also subjected to a TUNEL assay using the in situ cell death detection kit, TMR red (Roche Diagnostics GmbH). Cell death was expressed as the percentage of cells displaying typical nuclear apoptotic morphology or as the percentage of TUNEL-positive cells. In the EGFP/Gag-PKB or EGFP/HA-PKBT308D/S473D cotransfection experiments, treatments started after 36 h of transfection, and the percentage of apoptotic nuclei in each treatment was scored in the EGFP-positive cell population.
MTNs maintained for 48 h in the presence of muscle extract were washed and treated for an additional 24 h in basal medium containing BDNF (10 ng/ml) plus the indicated inhibitors. At the end of the treatments, cell survival was evaluated as described previously (Soler et al., 1998) (in the EGFP/Gag-PKB cotransfection experiments, only the EGFP-positive cells were counted). Alternatively, MTNs were fixed after 15 h of treatment and subjected to a TUNEL assay and to a Hoechst staining as described above.
Western blot and immunoprecipitation
Western blot and immunoprecipitation were performed as described (Egea et al., 2000). Antiphospho-Akt-Thr308 and antiphospho-Akt-Ser473 antibodies (New England Biolabs, Inc.), anti-pan ERK, anti-Shc, and anti-CaMKK (Transduction Laboratories), anti-PKB C-20 (Santa Cruz Biotechnology, Inc.), antiP-Tyr (clone 4G10) (Upstate Biotechnology), and anti-tubulin (Sigma-Aldrich) were used as suggested by the manufacturer. TrkA immunoprecipitation was performed using the antipanTrk antibody (203) as described previously (Becker et al., 1998).
PI 3-kinase assay
PI 3-kinase activity was measured in antiP-Tyr immunoprecipitates using L--phosphatidylinositol as substrate and [
-32P] ATP (Amersham Pharmacia Biotech) as described previously (Egea et al., 1999). Phosphorylated lipids were resolved in a thin layer chromatography, detected by autoradiography, and quantified in a PhosphorImager (Boehringer).
Ras activity assay
Activation of Ras was evaluated by a nonradioactive method using the GSTRBD (de Rooij and Bos, 1997) as described previously (Egea et al., 2000). Ras was detected using an antipan Ras antibody (Oncogene Research Products).
PKB kinase assay
At the end of treatments, cells were lysed in a buffer containing 1% Triton X-100, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonylfluoride, 10 mg/ml aprotinin, 2 mM benzamidine, 20 mg/ml leupeptin, 1 mM Na3VO4, 40 mM ß-glycerophosphate, and 25 mM NaF. PKB was immunoprecipitated with the C-20 anti-PKB antibody (Santa Cruz Biotechnology, Inc.) and protein GSeph-arose. Immunocomplexes were sequentially washed three times with lysis buffer, two times with 50 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, two times with 0.5 M LiCl, 100 mM Tris, pH 8.0, 1 mM EDTA, and once with 50 mM Tris, pH 7.4, 10 mM MgCl2. Kinase reaction was performed at room temperature for 30 min in a kinase buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl2, and 1 mM DTT supplemented with 2.5 mg of histone H2B (Boehringer) and 3 mCi of [-32P] ATP (Amersham Pharmacia Biotech). The reaction was stopped by adding SDS-PAGE sample buffer. Histone H2B was resolved by SDS-PAGE. Radioactive spots were detected by autoradiography and quantified in a PhosphorImager (Boehringer).
Fluorescence microscopy
PC12 cells or MTNs were plated into poliornitine-coated coverslips or into poliornitine/laminine-coated plates, respectively. Cells were transiently transfected with the EGFPPKBPH construct, and after 3648 h cells were starved of trophic support and treated as indicated. At the end of stimulations, the medium was removed, and the cells were fixed in 4% (wt/vol) paraformaldehyde in PBS (pH 7.4) for 30 min. Cells were washed twice with PBS and mounted using Vectashield (Vector Laboratories). Images were scanned on a Leica TCS 4D (PC12 cells) or on a ZEISS LSM 310 (MTNs) laser confocal microscope using an oil immersion objective of 63x. Percentage of cells displaying cell surface fluorescence was obtained counting 50100 cells per coverslip using an epifluorescence microscope with an objective of 40x.
Materials
BAPTA-AM was from Molecular Probes. W5, W7, TFP, and LY294002 were from Calbiochem-Novabiochem">Calbiochem-Novabiochem. BDNF was from Alomone Laboratories. All other reagents were from Sigma-Aldrich. AntipanTrk (203) and PC12 cells were a gift from D. Martin-Zanca (CSIC-Universidad de Salamanca, Salamanca, Spain). The construct encoding the GSTRBD fusion protein was a gift from F. McKenzie, (State University of New York, Stony Brook, NY). pSG5-Gag-PKB was a gift from J. Downward (Imperial Cancer Research Foundation, London, UK). pCMV5-HA-PKBT308D/S473D was a gift from D.R. Alessi (University of Dundee, Scotland, UK) and B.A. Hemmings (Friedrich Miescher Institute, Basel, Switzerland). 7S NGF was prepared in our laboratory as described previously (Mobley et al., 1976).
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Footnotes |
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Acknowledgments |
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This work was funded by the Comisión Interministerial de Ciencia y Tecnología through the Plan Nacional de Salud y Farmacia (SAF 2000-0164-C02-01), Telemarató de TV3 (edició 1997: Malalties Degeneratives Hereditàries), European Union Research, Transfer and Development program (QLG3-CT1999-00602). J. Egea is a postdoctoral fellow supported by the European Union RTD grant.
Submitted: 9 January 2001
Revised: 23 May 2001
Accepted: 29 June 2001
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References |
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Alessi, D.R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and B.A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15:65416551.[Abstract]
Andjelkovic, M., H.S. Suidan, R. Meier, M. Frech, D.R. Alessi, and B.A. Hemmings. 1998. Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells. Eur. J. Biochem. 251:195200.[Abstract]
Becker, E., R.M. Soler, V.J. Yuste, E. Gine, C. Sanz-Rodriguez, J. Egea, D. Martin-Zanca, and J.X. Comella. 1998. Development of survival responsiveness to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5, but not to nerve growth factor, in cultured motoneurons from chick embryo spinal cord. J. Neurosci. 18:79037911.
Bellacosa, A., J.R. Testa, S.P. Staal, and P.N. Tsichlis. 1991. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science. 254:274277.[Medline]
Burgering, B.M., and P.J. Coffer. 1995. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 376:599602.[Medline]
Coffer, P.J., and J.R. Woodgett. 1991. Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur. J. Biochem. 201:475481.[Abstract]
Comella, J.X., C. Sanz-Rodriguez, M. Aldea, and J.E. Esquerda. 1994. Skeletal muscle-derived trophic factors prevent motoneurons from entering an active cell death program in vitro. J. Neurosci. 14:26742686.[Abstract]
Conus, N.M., B.A. Hemmings, and R.B. Pearson. 1998. Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k. J. Biol. Chem. 273:47764782.
Crowder, R.J., and R.S. Freeman. 1998. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J. Neurosci. 18:29332943.
Currie, R.A., K.S. Walker, A. Gray, M. Deak, A. Casamayor, C.P. Downes, P. Cohen, D.R. Alessi, and J. Lucocq. 1999. Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1. Biochem. J. 337:575583.[Medline]
Czech, M.P. 2000. PIP2 and PIP3: complex roles at the cell surface. Cell. 100:603606.[Medline]
Datta, S.R., A. Brunet, and M.E. Greenberg. 1999. Cellular survival: a play in three Akts. Genes Dev. 13:29052927.
Davies, A.M. 1994. The role of neurotrophins in the developing nervous system. J. Neurobiol. 25:13341348.[Medline]
de Rooij, J., and J.L. Bos. 1997. Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras. Oncogene. 14:623625.[Medline]
D'Mello, S.R., K. Borodezt, and S.P. Soltoff. 1997. Insulin-like growth factor and potassium depolarization maintain neuronal survival by distinct pathways: possible involvement of PI 3-kinase in IGF-1 signaling. J. Neurosci. 17:15481560.
Dolcet, X., J. Egea, R.M. Soler, D. Martin-Zanca, and J.X. Comella. 1999. Activation of phosphatidylinositol 3-kinase, but not extracellular-regulated kinases, is necessary to mediate brain-derived neurotrophic factor-induced motoneuron survival. J. Neurochem. 73:521531.[Medline]
Dudek, H., S.R. Datta, T.F. Franke, M.J. Birnbaum, R. Yao, G.M. Cooper, R.A. Segal, D.R. Kaplan, and M.E. Greenberg. 1997. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science. 275:661665.
Egea, J., C. Espinet, and J.X. Comella. 1999. Calcium influx activates extracellular-regulated kinase/mitogen-activated protein kinase pathway through a calmodulin-sensitive mechanism in PC12 cells. J. Biol. Chem. 274:7585.
Egea, J., C. Espinet, R.M. Soler, S. Peiro, N. Rocamora, and J.X. Comella. 2000. Nerve growth factor activation of the extracellular signal-regulated kinase pathway is modulated by Ca2+ and calmodulin. Mol. Cell. Biol. 20:19311946.
Enslen, H., H. Tokumitsu, P.J. Stork, R.J. Davis, and T.R. Soderling. 1996. Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade. Proc. Natl. Acad. Sci. USA. 93:1080310808.
Espinet, C., X. Gomez-Arbones, J. Egea, and J.X. Comella. 2000. Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells. J. Neurosci. Methods. 100:6369.[Medline]
Finkbeiner, S., and M.E. Greenberg. 1996. Ca2+-dependent routes to Ras: mechanisms for neuronal survival, differentiation, and plasticity? Neuron. 16:233236.[Medline]
Fischer, R., J. Julsgart, and M.W. Berchtold. 1998. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase. FEBS Lett. 425:175177.[Medline]
Franklin, J.L., and E.M. Johnson. 1992. Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium. Trends Neurosci. 15:501508.[Medline]
Hack, N., H. Hidaka, M.J. Wakefield, and R. Balazs. 1993. Promotion of granule cell survival by high K1 or excitatory amino acid treatment and Ca2+/calmodulin-dependent protein kinase activity. Neuroscience. 57:920.[Medline]
Hallberg, B., M. Ashcroft, D.M. Loeb, D.R. Kaplan, and J. Downward. 1998. Nerve growth factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoinositide 3-OH kinase. Oncogene. 17:691697.[Medline]
Hidaka, H., and T. Tanaka. 1983. Naphthalenesulfonamides as calmodulin antagonists. Methods Enzymol. 102:185194.[Medline]
Holgado-Madruga, M., D.K. Moscatello, D.R. Emlet, R. Dieterich, and A.J. Wong. 1997. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc. Natl. Acad. Sci. USA. 94:1241912424.
Ikegami, K., and T. Koike. 2000. Membrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways. Brain Res. 866:218226.[Medline]
Jiang, H., and G. Guroff. 1997. Actions of the neurotrophins on calcium uptake. J. Neurosci. Res. 50:355360.[Medline]
Jones, P.F., T. Jakubowicz, F.J. Pitossi, F. Maurer, and B.A. Hemmings. 1991. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc. Natl. Acad. Sci. USA. 88:41714175.[Abstract]
Joyal, J.L., D.J. Burks, S. Pons, W.F. Matter, C.J. Vlahos, M.F. White, and D.B. Sacks. 1997. Calmodulin activates phosphatidylinositol 3-kinase. J. Biol. Chem. 272:2818328186.
Kaplan, D.R., and F.D. Miller. 2000. Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10:381391.[Medline]
Lewin, G.R., and Y.A. Barde. 1996. Physiology of the neurotrophins. Annu. Rev. Neurosci. 19:289317.[Medline]
Maruyama, T., T. Kanaji, S. Nakade, T. Kanno, and K. Mikoshiba. 1997. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J. Biochem. 122:498505.[Abstract]
Miller, T.M., M.G. Tansey, E.M. Johnson, and D.J. Creedon. 1997. Inhibition of phosphatidylinositol 3-kinase activity blocks depolarization- and insulin-like growth factor I-mediated survival of cerebellar granule cells. J. Biol. Chem. 272:98479853.
Mobley, W.C., A. Schenker, and E.M. Shooter. 1976. Characterization and isolation of proteolytically modified nerve growth factor. Biochemistry. 15:55435552.[Medline]
Moyers, J.S., P.J. Blan, J. Zhu, and C.R. Khan. 1997. Rad and Rad-related GTPases interact with calmodulin and calmodulin-dependent protein kinase II. J. Biol. Chem. 272:1183211839.
Obermeier, A., R. Lammers, K. Weismuller, G. Lung, J. Schlessinger, and A. Ullrich. 1993. Identification of Trk binding sites for SHC and phosphatidylinositol 3'-kinase and formation of a multimeric signaling complex. J. Biol. Chem. 268:2296322966.
Obermeier, A., I. Tinhofer, H.H. Grunicke, and A. Ullrich. 1996. Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation. EMBO J. 15:7382.[Abstract]
Pandiella-Alonso, A., A. Malgaroli, L.M. Vicentini, and J. Meldolesi. 1986. Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells. FEBS Lett. 208:4851.[Medline]
Park, E.K., S.I. Yang, and S.S. Kang. 1996. Activation of Akt by nerve growth factor via phosphatidylinositol-3 kinase in PC12 pheochromocytoma cells. Mol. Cell. 6:494498.
Philpott, K.L., M.J. McCarthy, A. Klippel, and L.L. Rubin. 1997. Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons. J. Cell Biol. 139:809815.
Rodriguez-Viciana, P., P.H. Warne, R. Dhand, B. Vanhaesebroeck, I. Gout, M.J. Fry, M.D. Waterfield, and J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 370:527532.[Medline]
Salinas, M., R. Lopez-Valdaliso, D. Martin, A. Alvarez, and A. Cuadrado. 2000. Inhibition of PKB/Akt1 by C2-ceramide involves activation of ceramide-activated protein phosphatase in PC12 cells. Mol. Cell. Neurosci. 15:156169.[Medline]
Soler, R.M., J. Egea, G.M. Mintenig, C. Sanz-Rodriguez, M. Iglesias, and J.X. Comella. 1998. Calmodulin is involved in membrane depolarization-mediated survival of motoneurons by phosphatidylinositol-3 kinase- and MAPK-independent pathways. J. Neurosci. 18:12301239.
Stephens, R.M., D.M. Loeb, T.D. Copeland, T. Pawson, L.A. Greene, and D.R. Kaplan. 1994. Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses. Neuron. 12:691705.[Medline]
Taules, M., A. Rodriguez-Vilarrupla, E. Rius, J.M. Estanyol, O. Casanovas, D.B. Sacks, E. Perez-Paya, O. Bachs, and N. Agell. 1999. Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization. J. Biol. Chem. 274:2444524448.
Ulrich, E., A. Duwel, A. Kauffmann-Zeh, C. Gilbert, D. Lyon, B. Rudkin, G. Evan, and D. Martin-Zanca. 1998. Specific TrkA survival signals interfere with different apoptotic pathways. Oncogene. 16:825832.[Medline]
Vaillant, A.R., I. Mazzoni, C. Tudan, M. Boudreau, D.R. Kaplan, and F.D. Miller. 1999. Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinaseAkt pathway to synergistically regulate neuronal survival. J. Cell Biol. 146:955966.
Vlahos, C.J., W.F. Matter, K.Y. Hui, and R.F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:52415248.
Weihl, C.C., G.D. Ghadge, S.G. Kennedy, N. Hay, R.J. Miller, and R.P. Roos. 1999. Mutant presenilin-1 induces apoptosis and downregulates Akt/PKB. J. Neurosci. 19:53605369.
Yamada, M., H. Ohnishi, S. Sano, A. Nakatani, T. Ikeuchi, and H. Hatanaka. 1997. Insulin receptor substrate (IRS)-1 and IRS-2 are tyrosine-phosphorylated and associated with phosphatidylinositol 3-kinase in response to brain-derived neurotrophic factor in cultured cerebral cortical neurons. J. Biol. Chem. 272:3033430339.
Yang, C., R.T. Watson, J.S. Elmendorf, D.B. Sacks, and J.E. Pessin. 2000. Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3L1 adipocytes. Mol. Endocrinol. 14:317326.
Yano, S., H. Tokumitsu, and T.R. Soderling. 1998. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature. 396:584587.[Medline]
Yao, R., and G.M. Cooper. 1995. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 267:20032006.[Medline]