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Keywords:

  • acid sphingomyelinase;
  • caveolae-related domains;
  • p75NTR;
  • phosphoinositide 3-kinase;
  • sphingomyelin;
  • Trk A

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75NTR neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75NTR-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75NTR. In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75NTR-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand–induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75NTR-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

Abbreviations used
BDNF

brain-derived neurotrophic factor

caPI 3-kinase

constitutively active phosphoinositide 3-kinase

CRDs

caveolae-related domains

dnPI 3-kinase

dominant-negative phosphoinositide 3-kinase

EGF

epidermal growth factor

IP

immunoprecipitation

MBS

MES-buffered saline

MBST

MBS + 1% Triton-X-100

MES

2-(4-morpholino) ethane sulfonic acid

NCMs

non-caveolar membranes

NGF

nerve growth factor

NRIF

NGF receptor interacting factor

NT-3

neurotrophin-3

PAS

protein A sepharose

PBS

phosphate buffered saline

pEGFP, enhanced

green fluorescent protein plasmid

PI 3-kinase

phosphoinositide 3-kinase

PtdIns-3-P

phosphatidylinositol-3-phosphate

SAPK

stress-activated protein kinases

SM

sphingomyelin

SMase

sphingomyelinase

TRAF

TNF receptor-associated factor

Neurotrophins are a family of growth factors that regulate the growth, differentiation, survival and death of distinct neuronal populations (Kaplan and Miller 1997). Each neurotrophin binds preferentially to a member of the Trk family of receptor-linked tyrosine kinases (Barbacid 1994); nerve growth factor (NGF) binds to Trk A, brain-derived neurotrophic factor (BDNF) binds to Trk B, and neurotrophin-3 (NT-3) binds to Trk C. In contrast, all the neurotrophins bind to the p75NTR neurotrophin receptor (Chao 1994).

In general, the Trk family of receptors regulate neuronal differentiation and survival via their tyrosine kinase activity (Klesse and Parada 1999). In contrast, a novel emerging paradigm is that p75NTR may serve as a potential neurotrophin responsive cell stress receptor (Barker 1998; Dobrowsky and Carter 2000). p75NTR may induce cell stress signals, in part, by the TRAF-dependent activation of NF-κB (Carter et al. 1996; Khursigara et al. 1999; Ye et al. 1999), coupling to a novel Zn2+ finger protein, NRIF (Casademunt et al. 1999), regulating Rho GTPase activity (Yamashita et al. 1999) or generating the sphingolipid metabolite ceramide (Dobrowsky et al. 1994; Brann et al. 1999).

Although the mechanisms by which p75NTR induces cellular responses remain to be fully elucidated, ceramide may be a critical component of p75NTR signaling in certain cell types. For example, p75NTR-dependent ceramide production may contribute to the induction of apoptosis in oligodendrocytes and cells of the inner ear (Cassacia-Bonnefil et al. 1996; Frago et al. 1998), the release of neurotransmitters in mesencephalic neurons (Blochl and Sirrenberg 1996) and is a necessary signal in regulating axonal growth in developing hippocampal neurons (Brann et al. 1999). These results suggest that p75NTR-dependent ceramide generation may activate very distinct signaling pathways depending upon the cellular context. The type of signaling pathways activated by p75NTR may be influenced, in part, by signals that emanate from Trk family members.

p75NTR and Trk family members are often co-expressed in many cell types. Given the opposing nature of the signals typically coupled to Trk activation (survival signals) versus p75NTR (apoptosis), it is predicted that crosstalk between Trk and p75NTR signaling pathways may regulate the cellular response to neurotrophins. Indeed, activation of Trk A blocked p75NTR-dependent death of oligodendrocytes (Yoon et al. 1998) and sympathetic neurons (Aloyz et al. 1998; Bamji et al. 1998). Interactions between Trk A-dependent growth and p75NTR-dependent death signals are of fundamental physiological significance in regulating the extent of target innervation by developing sympathetic neurons (Kohn et al. 1999). Thus, a growing body of evidence provides compelling support that signaling through Trk A-dependent pathways can negatively regulate p75NTR-dependent signal transduction.

Activation of Trk A can also inhibit sphingolipid signaling through p75NTR (Dobrowsky et al. 1995). In the absence of Trk receptors, all neurotrophins can induce SM hydrolysis in p75NTR-NIH-3T3 cells. However, in PC12 cells, which coexpress p75NTR and Trk A, NGF did not elicit SM hydrolysis unless Trk A tyrosine kinase activity was first inhibited with k252a. Therefore, we have been examining potential molecular mechanisms that regulate the crosstalk between Trk A tyrosine kinase and p75NTR-dependent sphingolipid signaling pathways. In this report, we provide evidence that ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase (EC 2.7.1.137)/phosphoinositide 3-kinase (PI 3-kinase) functions downstream of Trk A and negatively regulates p75NTR-dependent sphingolipid signaling through inhibition of an acidic sphingomyelin phosphodiesterase (EC 3.1.4.12)/acid sphingomyelinase (acid SMase) which localizes to caveolae-related domains.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Mouse 2.5S NGF was purchased from Harlan Bioproducts. NT-3 was from Alamone Laboratories. Anti-FLAG M2 monoclonal antibody, poly-l-lysine, phosphatidylinositol, genistein and wortmannin were purchased from Sigma Chemicals. LY294002 was from Calbiochem and k252a was obtained from Peptides International. Antibodies against the p85 subunit of PI 3-kinase and cavatellin were obtained from Upstate Biotechnology Inc. and Signal Transduction Labs, respectively. [3H]Choline chloride was purchased from American Radiolabeled Chemicals. [N-methyl-14C]Sphingomyelin was chemically synthesized by the base catalyzed methylation of N,N-dimethyl ceramidephosphoethanolamine with [14C]methyl iodide (Okazaki et al. 1994). The radiolabeled product was purified to homogeneity by silica gel column chromatography and preparative TLC. Various syntheses resulted in products with a specific activity of 40–80 mCi/mmol.

Cell lines, cDNA constructs and transfection procedure

PC12 cells were maintained in RPMI 1640 media containing 10% horse serum, 5% fetal bovine serum (FBS) and antibiotics. The cells were adhered to uncoated 10-cm tissue culture plates and were used for transfection experiments at about 60–70% confluency. PC12 cells were transiently transfected using the Transfast transfection reagent exactly as directed by the supplier (Promega). In brief, the cells were placed in 6 mL of serum free medium containing a 1 : 1 ratio of DNA to transfection agent (15 µg DNA:45 µL TransFast) for 1 h. The transfection medium was supplemented with 6 mL of prewarmed maintenance medium and after 24–48 h the cells were placed in RPMI containing 1% horse serum for 4 h prior to treatment with growth factors. In some experiments, pEGFP-N2, which codes for enhanced green fluorescent protein (Clontech), was used to assess for efficiency of the transfection. Routinely, the Transfast reagent resulted in > 70% of the adherent PC12 cells expressing the green fluorescent protein (data not shown).

Human acid SMase cDNA containing a carboxyl terminus FLAG epitope-tag was a kind gift from Dr R. Kolesnick (Schissel et al. 1998) and was subcloned into pCDNA 3.1. Myc-tagged kinase deficient (p110*Δkinase) and constitutively active (p110*) constructs of PI 3-kinase have been characterized previously (Hu et al. 1995). p110*Δkinase contains an inter-SH2 sequence of p85 which block association of this kinase negative p110 subunit with endogenous p85. To create a myc-tagged dominant negative kinase which could interact with endogenous p85, the inter-SH2 domain from the kinase-deficient p110*Δ kinase construct (Hu et al. 1995) was removed and the resulting dominant negative p110*Δkinase was subcloned into pcDNA 3.1 and sequenced.

Measurement of SM levels in PI 3-kinase transfected PC12 cells

PC12 cells were seeded onto poly-l-lysine-coated 6-well plates and were transfected with pCDNA 3.1 as a vector control, constitutively active PI 3-kinase cDNA or dominant negative PI 3-kinase cDNA. Following the transfection procedure, the cells were placed in medium containing 0.5 µCi/mL [3H]choline chloride for 2 days. After the labeling period, the cells were washed with PBS and placed in RPMI 1640 containing 1% horse serum prior to any further treatments. In some experiments, the cells were preincubated with the indicated concentration of k252a, wortmannin, LY294002 or vehicle for 1 h prior to neurotrophin stimulation. The cells were stimulated with NGF for 15 min or NT-3 for 12 min after which the medium was aspirated and the cells scraped into 2 mL of methanol. SM levels were measured as described previously (Bilderback et al. 1997, 1999).

Assay of acid SMase-associated PI 3-kinase activity

PC12 cells were grown in 10- or 15-cm dishes and were transfected either with empty vector or with FLAG-tagged acid SMase. Twenty-four hours after transfection, the cells were incubated overnight in RPMI 1640 containing 1% horse serum and stimulated with NGF or vehicle for 15 min. The medium was aspirated and the cells were washed with ice cold PBS containing 0.5 mm sodium orthovanadate. The cells were scraped into 1 mL of ice-cold immunoprecipitation buffer (IP) buffer (10 mm Tris–HCl, pH 7.4, 10 mm EDTA, 1 mm orthovanadate, 0.1 mm genistein, 1% Nonidet P-40, 60 mmβ-octylglucoside, 1 mm phenylmethylsulfonyl fluoride, and 10 µg/mL each of leupeptin, aprotinin and bestatin) and the cells were broken by sonication with three 15-s bursts. The lysate was centrifuged at 10 000 g for 5 min at 4°C and approximately 1.5 mg of protein from the supernatant was precleared with 2 µg of IgG coupled to protein A Sepharose (PAS) beads for 30 min. Following centrifugation of the immunocomplexes, the supernatant was recovered and incubated for 1 h at 4°C with 5 µg of the M2 monoclonal FLAG antibody. The FLAG-tagged acid SMase was immunoprecipitated by the addition of 5 µg of anti-mouse IgG and PAS beads for 1 h at 4°C.

The immunocomplexes were recovered by centrifugation and washed three times with 0.1 mm orthovanadate, 1% Nonidet P-40, 137 mm NaCl, 1 mm CaCl2, and 1 mm MgCl2 in 20 mm Tris, pH 7.4. The beads were then washed three times with 5 mm LiCl, 0.1 mm orthovanadate in 0.1 m Tris, pH 7.4, followed by three washes with TNE (10 mm Tris–HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA) (Milani et al. 1996). To assay lipid kinase activity, the beads were resuspended in 0.05 mL of TNE, phosphatidylinositol was added to a final concentration of 0.27 mg/mL and the reaction initiated by the addition of 30 µCi [γ-32P] ATP. After 10 min at 37°C, the reaction was quenched with 0.02 mL of 6 N HCl. The radiolabeled lipid phosphates were extracted by the addition of 0.16 mL of chloroform : methanol (1 : 1) and after recovery of the organic phase, the lipid phosphates were resolved by TLC using chloroform:methanol : water : ammonium hydroxide (60 : 47 : 11.3 : 2) as the developing solvent. The products were visualized by autoradiography, identified by co-migration with standards, and the radioactive bands quantitated by scintillation spectrometry.

Western blot analysis of immunoprecipitated P85 and FLAG-tagged acid SMase

Acid SMase was immunoprecipitated from PC12 cells using the anti-FLAG antibody as described above. The PAS beads were washed three times with MBST (25 mm MES, 150 mm NaCl, 5 mm EDTA, and 1% Triton) and the bound proteins were solubilized by boiling in sample buffer for 5 min. Following SDS-PAGE, the proteins were transferred to nitrocellulose and the membranes were probed with the PI 3-kinase p85 subunit antibody. Immunoreactive proteins were visualized using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. To detect the epitope-tagged acid SMase, the blot was stripped and probed a second time with monoclonal FLAG antibody. A recombinant human FLAG-tagged acid SMase protein standard was prepared by expression in Sf9 cells and was partially purified by affinity chromatography.

Assessment of acid SMase activity and protein association in CRDs

CRDs were isolated based upon their insolubility following non-ionic detergent extraction into ice cold MBST. Due to their low buoyant density, the detergent-insoluble CRDs were readily separated from detergent-soluble proteins by flotation in discontinuous sucrose gradients (Bilderback et al. 1997, 1999). Following centrifugation, 15 × 0.8 mL fractions were removed starting from the top of the gradient and total acid SMase activity assessed using 0.05 mL of each gradient fraction. Acid SMase activity was assayed as described using 0.2 mm[N-methyl-14C]SM (3000 dpm/nmol) (Liu and Anderson 1995). Alternatively, to examine protein association in CRDs, the cells were subjected to a non-detergent extraction using sodium carbonate as we and colleagues have described previously (Song et al. 1996; Bilderback et al. 1999). Following flotation of the CRDs in the discontinuous sucrose gradients, the membranes were isolated by pooling fractions 4–6 (caveolae-related domains) and fractions 11–15 (non-caveolar membranes), diluting the pooled fractions with MBS, and recovering the membranes by centrifugation at 100 000 g for 1 h at 4°C. The isolated membranes were solubilized in IP buffer and immunoprecipitations were performed as described above. In some experiments, acid SMase activity was measured following immunoprecipitation of FLAG-tagged enzyme from control and NGF-treated cells. The washed immunoprecipitates were resuspended in 0.05 mL of acid SMase assay buffer and the reaction initiated by the addition of 0.05 mL of substrate in the same assay buffer. All enzyme activities were linear with respect to time and protein and no more than 10% of the substrate was hydrolyzed.

Statistical analysis

Statistical differences within a data set were determined using a one-way anova. Differences between group means were determined using a Student–Newman–Keuls analysis. Means were deemed significantly different at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Trk A activation of PI 3-kinase inhibits p75NTR-dependent SM hydrolysis

Similar to our previous results (Dobrowsky et al. 1995), treatment of PC12 cells with 10 ng/mL NT-3 but not 100 ng/mL NGF induced a significant decrease in SM levels (Fig. 1). It is important to note that although these studies were performed at a single time point, we have characterized extensively the dose-dependence and kinetics of neurotrophin-induced SM turnover in PC12 cells (Dobrowsky et al. 1995; Bilderback et al. 1997, 1999). We have also shown that changes in SM levels based upon metabolic labeling correspond closely to similar changes in SM mass (Dobrowsky et al. 1994, 1995). Thus, this and subsequent data were obtained at an optimized dose and time where neurotrophin-induced SM hydrolysis is typically maximal.

image

Figure 1. (a) NGF induces SM hydrolysis through p75NTR after pharmacologic inhibition of PI 3-kinase. PC12 cells were metabolically labeled with [3H]choline for 2 days, washed in ice cold PBS and placed in RPMI 1640 medium containing 1% horse serum for 4 h at 37°C. The cells were then incubated with vehicle, 200 nm wortmannin or 5–20 µm LY294002 for 1 h prior to treatment with PBS, NGF (100 ng/mL for 15 min) or NT-3 (10 ng/mL NT-3 for 12 min). The medium was aspirated, the cells scraped into 2 mL of methanol and total lipids were extracted. [3H]SM levels were determined as described in Materials and methods and are expressed as a percent of control. The data shown are mean ± SE from 3 to 4 experiments performed in triplicate. * and § indicate significant difference versus control or NGF, respectively. (b) LY294002 does not inhibit NGF-induced Trk A autophosphorylation. PC12 cells were incubated with 200 nm k252a or 20 µm LY294002 for 1 h prior to stimulation with 100 ng/mL NGF for 15 mins. Trk A was immunoprecipitated and the proteins were resolved by SDS-PAGE. After transfer to nitrocellulose, the blot was first probed with an anti-phosphotyrosine antibody (upper panel) then stripped and re-probed with a pan-Trk antibody (lower panel).

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To determine if PI 3-kinase may function downstream of Trk A to inhibit p75NTR-mediated SM hydrolysis, we initially used two pharmacological inhibitors of PI 3-kinase, wortmannin and LY294002. In the absence of wortmannin pretreatment, NGF did not induce SM hydrolysis in PC12 cells. However, pretreatment of the cells with 200 nm wortmannin led to a modest but significant decrease in SM levels following NGF stimulation (Fig. 1). Though wortmannin irreversibly inhibits PI 3-kinases, it is not strictly specific for this enzyme (Fruman et al. 1998). Therefore, we also examined the effect of a reversible inhibitor of PI 3-kinase, LY294002, on NGF-induced SM hydrolysis. Similar to wortmannin, pretreatment of the cells with 5–20 µm LY294002 also induced a significant dose-dependent decrease in SM levels (Fig. 1). Importantly, LY294002 treatment alone had no effect on SM levels (see Fig. 3c).

image

Figure 3. Expression of caPI 3-kinase inhibits p75NTR-mediated SM hydrolysis. (a) PC12 cells were transfected transiently with either empty vector or caPI 3-kinase. The cells were treated with PBS or 100 ng/mL NGF for 15 min and cell lysates were prepared. caPI 3-kinase was immunoprecipitated using an antibody against the myc epitope tag and the lipid kinase activity of the immunocomplexes was assessed using an in vitro kinase assay. The migration position of authentic PtdIns-3-P is indicated by the arrow. (b) Cells transfected with vector only or caPI 3-kinase were metabolically labeled with [3H]choline as described in Materials and methods. The cells were treated with vehicle or 200 nm k252a for 1 h prior to treatment with PBS, NGF or NT-3. SM levels were determined and expressed as a percent of control. (c) Cells were prepared as above except that some cells were also treated with 20 µm LY294002 for 1 h prior to treatment with PBS or NGF. SM levels were determined and expressed as a percent of control. All data are the mean ± SE and are from 3 to 5 experiments performed in triplicate. * and § indicate significant difference versus control or NGF, respectively.

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Although LY294002 inhibits PI 3-kinase in vitro with an IC50 of about 1–2 µm, concentrations of LY294002 from 10 to 100 µm are often necessary to inhibit PI 3-kinase in intact cells (Crowder and Freeman 1998). Indeed, we observed that 20 µm LY294002 inhibited the NGF induced stimulation of PI 3-kinase activity by > 95% but did not block NGF-induced Trk A autophosphorylation (Fig. 1b). Therefore, the effect of LY294002 in enabling NGF to induce SM hydrolysis is due to inhibition of PI 3-kinase activity and not the inactivation of Trk A by the drug.

If activation of PI 3-kinase by Trk A is critical for silencing p75NTR-mediated SM hydrolysis in PC12 cells, then a PI 3-kinase inhibitor should have no effect on SM hydrolysis induced by neurotrophins which do not activate Trk A. NT-3 does not activate Trk A nor PI 3-kinase in PC12 cells (data not shown) (Belliveau et al. 1997). Treatment of PC12 cells with NT-3 induced a significant decrease in SM levels but this hydrolysis was insensitive to treatment with LY294002 (Fig. 1). Together, these pharmacological data support that Trk A activation of PI 3-kinase is a negative regulator of p75NTR-dependent sphingolipid signaling.

NGF induces SM hydrolysis in cells expressing a dominant negative PI 3-kinase

Since pharmacological approaches always invoke a concern regarding drug specificity, a molecular approach to further establish the involvement of PI 3-kinase in regulating p75NTR-mediated SM hydrolysis was taken. Expression of p110 Δkinase is recognized to act as a dominant negative PI 3-kinase (dnPI 3-kinase) and inhibits Trk A mediated cell survival (Crowder and Freeman 1998).

Transient transfection of PC12 cells with the dnPI 3-kinase inhibited basal PI 3-kinase activity and blocked NGF-induced activation of endogenous PI 3-kinase by about 40% (Fig. 2a). Although prolonged expression of dnPI 3-kinase induces cell death (Crowder and Freeman 1998), we observed no significant difference in viability between cells receiving empty vector or dnPI 3-kinase after 48 h. Further, expression of the dnPI 3-kinase had no effect on NGF-induced Trk A autophosphorylation (data not shown).

image

Figure 2. Expression of dnPI 3-kinase reverses Trk A inhibition of p75NTR-dependent SM hydrolysis. (a) PC12 cells were transfected transiently with either empty vector or dnPI 3-kinase. The cells were treated with PBS or 100 ng/mL NGF for 15 min and lipid kinase activity immunoprecipitated with an antibody against the p85 subunit of PI 3-kinase. Lipid kinase activity of the immunoprecipitates was determined using an in vitro kinase assay. The migration position of authentic PtdIns-3-P is indicated by the arrow. (b) Cells transfected with vector only or dnPI 3-kinase were metabolically labeled with [3H]choline as described in Materials and methods. The cells were treated with vehicle or 200 nm k252a for 1 h prior to treatment with PBS or 100 ng/mL NGF for 15 min. SM levels were quantitated and expressed as a percent of control. The data are the mean ± SE from 2 experiments performed in triplicate. * and § indicate significant difference versus control or NGF, respectively.

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If Trk A activation of PI 3-kinase inhibits p75NTR-mediated SM hydrolysis, then expression of dnPI 3-kinase should mimic pharmacologic inhibition of this lipid kinase. Similar to our previous reports (Dobrowsky et al. 1995; Bilderback et al. 1999), inhibition of Trk A tyrosine kinase with k252a (Fig. 1b) was necessary for NGF to induce SM hydrolysis in PC12 cells (Fig. 2b, closed bars). In contrast, NGF significantly decreased SM levels in PC12 cells expressing the dnPI 3-kinase even in the absence of Trk A inhibition with k252a (Fig. 2b, open bars). These results provide molecular support that activation of PI 3-kinase is required to inhibit SM turnover mediated through p75NTR.

Expression of a constituitively active PI 3-kinase prevents neurotrophin-induced SM hydrolysis

p110* is a constitutively active PI 3-kinase (caPI 3-kinase) mutant in which the inter-SH2 domain of the p85 subunit is ligated to the amino terminus of the p110 subunit (Hu et al. 1995). We hypothesized that expression of caPI 3-kinase would increase the basal level of PI 3-kinase activity and inhibit p75NTR-dependent SM hydrolysis regardless of ligand or the activation state of Trk A. Expression of caPI 3-kinase in PC12 cells was very efficient and resulted in a 13-fold increase in basal PI 3-kinase activity (Fig. 3a). Therefore, PC12 cells were transiently transfected with either empty vector or caPI 3-kinase cDNA and stimulated with NGF in the absence or presence of k252a. In cells transfected with the empty vector, NGF could only induce SM hydrolysis if Trk A was first inactivated with k252a (Fig. 3b, closed bars). However, expression of the caPI 3-kinase blocked NGF-induced SM hydrolysis even when Trk A activity was inhibited with k252a (Fig. 3b, open bars).

Next, the effect of caPI 3-kinase expression on SM hydrolysis induced by neurotrophins which only signal through p75NTR was examined. NT-3 induced a significant decrease in SM levels in PC12 cells transfected with empty vector (Fig. 3b). However, this hydrolysis was insensitive to k252a since NT-3 does not activate Trk A in PC12 cells (Belliveau et al. 1997). Importantly, NT-3 did not induce SM hydrolysis in PC12 cells expressing caPI 3-kinase (Fig. 3b). This result indicates that constitutive activation of PI 3-kinase can inhibit p75NTR-dependent SM hydrolysis and bypass the need to first activate Trk A.

It is possible that the high level of expression of the caPI 3-kinase was inhibiting p75NTR-dependent SM hydrolysis in a non-specific manner unrelated to the overall increase in lipid kinase activity. To investigate this possibility, we inhibited the activity of the caPI 3-kinase with LY29004. Similar to the results in Fig. 1, NGF decreased SM levels following LY294002 pretreatment of cells transfected with empty vector (Fig. 3c, closed bar). Significantly, LY294002 reversed the inhibitory effect of caPI 3-kinase expression on NGF-induced SM hydrolysis (Fig. 3c, compare open bars NGF + k252a with NGF + LY294002). Taken together, our pharmacological and molecular data strongly suggest that PI 3-kinase is a negative regulator of SM hydrolysis by p75NTR. These results portend the next question, what is the mechanism by which PI 3-kinase inhibits sphingolipid signaling?

Acid SMase is present in CRDs from PC12 cells

We have reported previously that p75NTR-dependent SM hydrolysis occurs in caveolae-related domains (CRDs) present in the plasma membrane of PC12 cells (Bilderback et al. 1997, 1999). Since acidic and neutral SMases are the only known mammalian enzymes that metabolize SM, these results suggested that a SMase may reside in CRDs and be a potential target for inhibition of PI 3-kinase. Indeed, a pool of acid SMase localizes to caveolae from fibroblasts (Liu and Anderson 1995; Zundel et al. 2000). Although caveolae and CRDs share a similar lipid composition, they differ in the expression of certain proteins (Dobrowsky 2000). CRDs contain the protein cavatellin but lack caveolin-1; caveolin-1 expression is required for the formation of caveolae proper (Volonte et al. 1999). We also detected a pool of acid SMase (Fig. 4) but not neutral SMase (data not shown) within CRDs of PC12 cells. Although the amount of acid SMase which localized to CRDs was small (Fig. 4a, open circles), the specific activity of this pool of enzyme was high (Fig. 4b, open circles) since CRDs contain less than 1% of total cellular protein (Fig. 4a, open squares).

image

Figure 4. Heterologously expressed FLAG-tagged human acid SMase is targeted to similar membrane fractions as endogenous enzyme. Four 10-cm dishes of PC12 cells were transiently transfected with FLAG-tagged human acid SMase cDNA or empty vector. After 48 h, the cells were scraped into ice cold MBST and CRDs were prepared as described in Materials and methods. Fractions of 0.8 mL were collected starting from the top of the gradient and the acid SMase activity was assessed. (a) Distribution of the protein (□, ▪) and acid SMase activity (○,●) in gradient fractions from cells transfected with empty vector (open symbols) or FLAG-tagged acid SMase (closed symbols). (b) The specific activity of acid SMase in gradient fractions isolated from cells transfected with empty vector (○) or FLAG-tagged acid SMase (●).

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To permit an analysis of potential protein interactions between acid SMase and PI 3-kinase, we chose to express a FLAG-tagged form of human acid SMase in PC12 cells. This approach was taken since antibodies capable of immunoprecipitating native acid SMase are not readily available. We first determined if the FLAG-tagged human acid SMase was efficiently expressed and had the same cellular localization as the endogenous enzyme. PC12 cells were transfected transiently with the epitope-tagged acid SMase, the cells subjected to a detergent extraction, and CRDs isolated from other cellular membranes by centrifugation through a discontinuous sucrose gradient. Similar to the distribution of the endogenous activity, most of the FLAG-tagged acid SMase was recovered in the non-caveolae membrane fraction of the gradient with a small pool also being targeted to CRDs (Fig. 4a, closed circles). Following transfection, the specific activity of acid SMase increased about two- to threefold in all membrane pools (Fig. 4b, closed circles). We avoided gross overexpression since dramatic elevation of endogenous ceramide levels in PC12 cells may lead to apoptosis (Hartfield et al. 1997). Importantly, these data indicate that the epitope-tagged enzyme targets to cellular membranes similar to the endogenous enzyme.

NGF-induced association of PI 3-kinase correlates with decreased acid SMase activity

Although several classes of PI 3-kinase exist, the class I signal-activated form consists of a p85 regulatory subunit and a p110 catalytic subunit (Rameh and Cantley 1999). In view of the inhibitory effect of PI 3-kinase on p75NTR-dependent sphingolipid signaling, we hypothesized that ligand treatment may induce an interaction of PI 3-kinase with acid SMase leading to an inhibition of the type C phospholipase activity of the SMase. Therefore, PC12 cells were transfected transiently with empty vector or the FLAG-tagged acid SMase cDNA and treated with vehicle or 100 ng/mL NGF for 15 min. Cell lysates were prepared and the FLAG-tagged acid SMase was immunoprecipitated from the whole cell lysate. The experiments depicted in Fig. 5 and Table 1 were performed in parallel cultures to allow for the measurements of protein association, PI 3-kinase activity and acid SMase activity following immunoprecipitation. In the absence of NGF treatment, the p85 subunit of PI 3-kinase did not co-immunoprecipitate with the epitope-tagged acid SMase, indicating that little constitutive association occurs between these proteins (Fig. 5a). However, NGF treatment for 15 min resulted in the co-immunoprecipitation of the p85 subunit of PI 3-kinase with acid SMase. Re-probing the same blot with an anti-FLAG antibody revealed that similar amounts of the epitope-tagged enzyme were immunoprecipitated from control and NGF-treated cells. Thus, the association of PI 3-kinase was not due to differences in the expression of acid SMase (Fig. 5a). Moreover, PC12 cells receiving empty vector showed no specific immunoreactive band for either p85 or acid SMase following immunoprecipitation with the FLAG antibody (data not shown).

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Figure 5. NGF induces the association of PI 3-kinase with acid SMase. PC12 cells were transfected with FLAG-tagged human acid SMase and after 24 h the cells were placed in RPMI 1640 containing 1% horse serum and maintained overnight. The cells were then treated with vehicle or 100 ng/mL NGF for 15 min and scraped into IP buffer. (a) Acid SMase was immunoprecipitated with an anti-FLAG antibody and the presence of the p85 subunit of PI 3-kinase associated with the immunoprecipitate determined by immunoblot analysis. The blot was stripped and reprobed to determine the levels of FLAG-tagged acid SMase. Protein standards for the p85 subunit of PI 3-kinase and recombinant human acid SMase (aSMase) are indicated. (b) PI 3-kinase from control and NGF treated cells was immunoprecipitated with an antip85 or anti-FLAG antibody. Lipid kinase activity was determined as described in Materials and methods.

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Table 1.   Association of PI 3-kinase with acid SMase correlates with decreased phospholipase activity
 Enzyme activity (dpm)
 PI 3-kinaseAcid Smase
100 ng/mL NGF++
  1. PC12 cells were transfected with FLAG-tagged human acid SMase and after 24 h the cells were placed in RPMI 1640 containing 1% horse serum and maintained overnight. The cells were treated with vehicle or 100 ng/mL NGF for 15 min and scraped into IP buffer. PI 3-kinase was immunoprecipitated with an anti-p85 antibody and lipid kinase activity was determined. Acid SMase was immunoprecipitated with an anti-FLAG antibody. The enzymatic activity of the immunoprecipitated acid SMase and any associated PI 3-kinase was determined as described in Materials and methods. The data for PI 3-kinase activity are from the experiment in Fig. 5(b). The data showing the effect of NGF on immunoprecipitated acid SMase activity are the mean ± SE from 3 experiments performed in duplicate. N.D., not detected.

IP antibody
 FLAG31235 6581508 ± 50769 ± 59
 p8520854 180N.D.N.D.

To address whether NGF also increased the activity of the PI 3-kinase which associated with acid SMase, cell lysates from control and NGF treated cells were immunoprecipitated with the FLAG antibody and the immunoprecipitates assayed for PI 3-kinase activity. Consistent with the absence of significant constitutive association of the p85 subunit of PI 3-kinase with acid SMase, little lipid kinase activity was associated with FLAG acid SMase from control cells (Fig. 5b and Table 1). However, NGF significantly increased the PI 3-kinase activity that coimmunoprecipitated with FLAG-tagged acid SMase. The association of PI 3-kinase with acid SMase also correlated with about a 50% decrease in the immunoprecipitated acid SMase activity (Table 1). These data suggest that the association of PI 3-kinase with acid SMase might contribute to the in vivo inhibitory effect of PI 3-kinase on SM metabolism (Figs 1–3).

Association of PI 3-kinase with acid SMase is not sufficient to inhibit phospholipase activity

Our initial pharmacological results indicated that PI 3-kinase activity was necessary for the inhibition of NGF-induced SM hydrolysis. Since NGF also induced the association of PI 3-kinase with acid SMase, we examined whether the stimulation of lipid kinase activity was also necessary for this interaction. To approach this question, PC12 cells were transfected with FLAG-tagged acid SMase and pretreated with vehicle or LY294002 to inhibit PI 3-kinase activity. We hypothesized that if increased PI 3-kinase activity was necessary for its association with acid SMase, then inhibition of the lipid kinase activity should block their NGF-induced interaction. In agreement with our in vivo data on SM hydrolysis (Figs 1 and 3c), LY294002 also abolished the inhibitory effect of PI 3-kinase on the immunoprecipitated acid SMase activity (Fig. 6). However, LY294002 had no effect on the NGF-induced association of the p85 subunit of PI 3-kinase with acid SMase. These data strongly support that PI 3-kinase activity is necessary for inhibiting acid SMase but that other factors regulate the association of the p85 subunit of this enzyme with acid SMase.

image

Figure 6. NGF stimulation of PI 3-kinase activity is necessary for inhibition of acid SMase activity but not for association with p85. PC12 cells were transfected with FLAG-tagged human acid SMase and after 24 h the cells were placed in RPMI 1640 containing 1% horse serum and maintained overnight. The cells were then pretreated with vehicle or 20 µm LY294002 for 1 h prior to treatment with PBS or 100 ng/mL NGF for 15 min. Acid SMase was immunoprecipitated with an anti-FLAG antibody and the presence of the p85 subunit of PI 3-kinase associated with the immunoprecipitates determined by immunoblot analysis. The blot was stripped and reprobed to determine the levels of FLAG-tagged acid SMase. The effect of LY294002 treatment on the immunoprecipitated acid SMase activity from sister cultures are shown below the gel and are mean ± SE from three experiments.

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The association of PI 3-kinase with acid SMase is specific for NGF

The activation of PI 3-kinase also occurs in response to growth factors such as epidermal growth factor (EGF) (Rameh and Cantley 1999). We have demonstrated previously that p75NTR-dependent sphingolipid signaling was inhibited specifically by NGF but not by activation of the EGF receptor, which is also a receptor-linked tyrosine kinase (Dobrowsky et al. 1995). These results suggest that the association of acid SMase with PI 3-kinase may also be specific for NGF. Indeed, NGF, but not EGF, induced the association of activated PI 3-kinase with acid SMase (Fig. 7). As expected, both NGF and EGF increased PI 3-kinase activity immunoprecipitated with an anti-p85 antibody from cells transfected with either empty vector (lower panel, lanes 4 and 6) or acid SMase (upper panel, lanes 4 and 6). However, PI 3-kinase activity only coimmunoprecipitated with acid SMase from NGF (upper panel, lane 3) but not EGF (upper panel, lane 5) treated cells. Together, these results strongly suggest that the association of PI 3-kinase with acid SMase requires NGF-specific signals or adapter proteins.

image

Figure 7. The association of PI 3-kinase with acid SMase is specific for NGF-PC12 cells were transfected with empty vector or FLAG-tagged human acid SMase. After 24 h, the cells were placed in RPMI 1640 containing 1% horse serum and maintained overnight. The cells were treated with vehicle or 100 ng/mL NGF for 15 min or 10 ng/mL EGF for 5 min and scraped into IP buffer. The lysates were then incubated with an antip85 or an anti-FLAG antibody and the proteins immunoprecipitated as described in Materials and methods. The associated lipid kinase activity of the immunoprecipitates was determined using an in vitro kinase assay.

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NGF-induced association of PI 3-kinase with acid SMase localizes to CRD

Signaling through both Trk A (Huang et al. 1999) and p75NTR (Bilderback et al. 1997) localizes to CRDs in PC12 cells, suggesting that these specialized lipid domains may be an important site for crosstalk between neurotrophin-signaling pathways. To examine if the NGF-induced association of PI-3 kinase with acid SMase occurred specifically in these membrane domains, we isolated CRDs and non-caveolar membranes (NCMs) from vehicle and NGF-treated cells. As a marker for the presence of CRDs, we examined the expression of cavatellin, a protein marker which is enriched in these membrane domains (Volonte et al. 1999). Indeed, cavatellin was highly enriched in CRDs vs. NCMs from PC12 cells when examined at equal protein levels (Fig. 8b).

image

Figure 8. The association of the p85 subunit of PI 3-kinase with acid SMase occurs in CRDs. (a) PC12 cells were transfected with empty vector or FLAG-tagged human acid SMase. After 24 h, the cells were placed in RPMI 1640 containing 1% horse serum and maintained overnight. The cells were treated with vehicle or 100 ng/mL NGF and scraped into 2 mL of 0.5 m Na2CO3. CRDs were separated from non-caveolar membranes (NCMs) by flotation in discontinuous sucrose gradients. The gradient fractions corresponding to CRDs (4–7) and NCMs (11–15) were pooled and the membranes sedimented by centrifugation. Acid SMase was immunoprecipitated following solubilization of the membranes in IP buffer and incubation with an agarose-conjugated anti-FLAG antibody for 2 h at 4°C. The beads were washed three times with MBST, the associated proteins separated by SDS-PAGE and the presence of p85 determined by immunoblot analysis. The blot was then stripped and re-probed with the FLAG antibody. (b) CRDs from PC12 cells are enriched in cavatellin. CRDs and NCMs were isolated as above and the proteins were resolved by SDS-PAGE. Equal amounts of protein (5 µg) from both membrane fractions were resolved by SDS-PAGE and transferred to nitrocellulose. The presence of cavatellin was determined by immunoblot analysis. The cavatellin standard is from a cell lysate supplied with the antibody for use as a positive control.

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Although the bulk of acid SMase activity localized to NCMs (Fig. 4), NGF did not induce an association between p85 and acid SMase from these membranes (Fig. 8a). In contrast, PI 3-kinase co-immunoprecipitated with acid SMase from the CRDs isolated from NGF treated cells. These results support that the interaction of acid SMase with PI 3-kinase occurs primarily within these specialized lipid domains.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this report we provide a molecular basis for our previous observation that NGF-induced Trk A activation inhibited p75NTR-mediated SM hydrolysis (Dobrowsky et al. 1995). Based upon pharmacological and molecular evidence, our data strongly suggest that PI 3-kinase is a critical regulator of crosstalk between Trk A tyrosine kinase and p75NTR-dependent sphingolipid signaling pathways through inhibition of acid SMase.

Trk A may inhibit p75NTR-mediated signaling directly or indirectly. Although we can not definitively rule out the former possibility, SM hydrolysis was rather equivalent following pharmacologic inhibition of PI 3-kinase or Trk A (Figs 1–3). Since LY294002 had no effect on the extent of Trk A activation by NGF, we conclude that Trk A does not directly inhibit p75NTR-dependent SM hydrolysis through an inhibitory tyrosine phosphorylation of acid SMase. Although our results leave open the possibility that acid SMase may be a target for tyrosine phosphorylation by Trk A, it is likely that this post-translational modification alone, if it occurs at all, has little impact on activity. Indeed, acid SMase activity is relatively insensitive to compounds such as sodium vanadate and genistein that regulate the levels of protein tyrosine phosphorylation (Nikolova-Karakashian et al. 1997).

Trk A activates numerous signaling molecules involved in regulating cell growth and differentiation (Klesse and Parada 1999). Since we have not directly examined whether all these molecules may regulate p75NTR-dependent sphingolipid signaling, it can be argued that LY294002 is non-specifically uncoupling activated Trk A from other signaling components which may inhibit acid SMase. However, caPI 3-kinase inhibited NT-3-induced SM hydrolysis in the absence of any Trk A activation (Fig. 2c), suggesting that alternative signals from Trk A are not necessary to inhibit this aspect of p75NTR signaling. Moreover, in cells expressing the dnPI 3-kinase, Trk A can undergo NGF-induced autophosphorylation and stimulate other signaling molecules. If these alternative signals contribute to inhibiting SM hydrolysis, the magnitude of NGF-induced SM hydrolysis should be less than that seen in cells that had all Trk A-dependent signals blocked with k252a. However, the level of NGF-induced SM hydrolysis in cells expressing dnPI 3-kinase was identical to that from cells which had Trk A inhibited with k252a (Fig. 3b). Collectively, these results support that PI 3-kinase is sufficient to inhibit SM hydrolysis and provide indirect support that other Trk A-dependent signaling molecules play little if any role in inhibiting p75NTR-dependent sphingolipid signaling.

Similar to previous results from human fibroblasts (Liu and Anderson 1995; Zundel et al. 2000), we identify that a pool of acid SMase of high specific activity is localized within CRDs (Figs 4 and 8). This localization is congruous with our previous reports that p75NTR-dependent SM hydrolysis also localized to CRDs in PC12 cells (Bilderback et al. 1997).

A surprising outcome of our study was the finding that NGF treatment enhanced the amount of the p85 subunit and lipid kinase activity which co-immunoprecipitated with acid SMase. Importantly, the co-immunoprecipitation of PI 3-kinase with acid SMase correlated with a 50% decrease in acid sphingomyelinase activity and occurred specifically in CRDs isolated from NGF-treated cells. Interestingly, the amount of acid SMase that was associated with the CRDs increased slightly following NGF treatment. Since no differences in the expression of acid SMase was apparent after immunoprecipitation from whole cell lysates (Figs 5 and 6), these results raise the possibility that some acid SMase may be recruited to CRDs following NGF treatment. Additionally, we observed that about 60% of the total PI 3-kinase activity immunoprecipitated from NGF-treated cells co-immunoprecipitated with acid SMase (Table 1). Given that the amount of acid SMase in the CRDs appears rather low, this extent of association would seem high. Since we are exogenously expressing acid SMase in this compartment, the amount of association with endogenous PI 3-kinase may be greater than would occur in the absence of the moderate over-expression.

Localization of crosstalk between neurotrophin receptor signaling pathways to CRDs would agree with a recent report indicating that NGF-induced Trk A autophosphorylation and downstream signaling occurs primarily within CRDs from PC12 cells (Huang et al. 1999). Indeed, an intact CRD domain was necessary for functional signal transduction since disruption of CRDs with cholesterol binding drugs inhibited NGF-induced Trk A autophosphorylation (Huang et al. 1999). Further, CRDs may be important sites for interactions between p75NTR and other Trk family members in neurons since both p75NTR and Trk B localize to CRDs obtained from synaptosomal preparations (Wu et al. 1997).

At this point, we can not ascertain if the NGF-induced association of PI 3-kinase with acid SMase is a prerequisite for the inhibition of sphingomyelinase activity. However, mere association of PI 3-kinase with acid SMase was not sufficient to inhibit sphingomyelinase activity. Indeed, LY294002 did not block ligand-induced association of PI 3-kinase with acid SMase but did reverse the inhibitory effect of PI 3-kinase on sphingomyelinase activity (Fig. 6) Further, inhibition of SM hydrolysis by caPI 3-kinase was also blocked by LY294002 (Fig. 3c). Together, these data strongly suggest that the lipid kinase activity of the enzyme is necessary to inhibit acid SMase activity. However, we have been unable to demonstrate an association between acid SMase and the caPI 3-kinase (likely due to the presence of the inter-SH2 domain which obscures the ability of the p110* subunit to associate with p85 (Hu et al. 1995). These data would argue that direct association of PI-3-kinase might not be necessary for inhibition of acid SMase. Alternatively, due to the high activity of the caPI 3-kinase (Fig. 3a), it may be producing substantial amounts of phosphatidylinositol-3-phosphate that strongly inhibits both endogenous and recombinant acid SMase (Dowbrowsky et al., unpublished data). Alternatively, PI 3-kinase also has an intrinsic ser/thr kinase activity which may use acid SMase as a substrate following association leading to diminished sphingomyelinase activity. Again, the high basal activity of the caPI 3-kinase may negate the need for association to induce this post-translational modification.

Although the epitope-tagged acid SMase was moderately over-expressed, the possibility exists that its association with PI 3-kinase may be non-specific. In our view, two pieces of evidence argue against this possibility. First, the association of PI 3-kinase with acid SMase was not constitutive but occurred only after stimulation of the cells with NGF; this also rules out a potential non-specific association of p85 with the FLAG epitope tag. Second, although EGF effectively activated PI 3-kinase, it was ineffective at inducing the association of PI 3-kinase with acid SMase. Together, these data indicate that the interaction of PI 3-kinase with acid SMase is specific, but they do not provide any insight into whether this NGF-induced protein–protein interaction is direct or may require additional adapter proteins.

The p85 subunit of PI 3-kinase possesses two src homology 2 (SH2) domains (Fruman et al. 1998). It is known that the interaction of the SH2 domains of the p85 subunit with tyrosine phosphorylated residues in YXXM sequences on target proteins leads to association and increased lipid kinase activation (Backer et al. 1992; Fruman et al. 1998). Acid SMase does possess a YEAM motif, which after tyrosine phosphorylation could serve as a recognition site for the SH2 domains present within the p85 subunit of PI 3-kinase. Although acid SMase is constitutively phosphorylated, we have not detected significant increases in tyrosine phosphorylation after NGF treatment. Site-directed mutagenesis of the YXXM motif of acid SMase will help address the potential role of this sequence in directing interactions with p85.

The p85 subunit of PI 3-kinase also contains a SH3 domain that may interact with proline-rich motifs (Fruman et al. 1998). Known SH3-binding proteins contain at least one PXXP motif (Sparks et al. 1996). Interestingly, acid SMase contains two putative minimal consensus sequences that may interact with SH3 domains. These sequences are in a proline rich hinge domain that links the saposin-like N-terminal activation domain with the C-terminal catalytic domain. Alternatively, adapter/docking proteins such as Shc, FRS2α, or Gab1 may be involved in regulating this NGF-induced interaction.

Both biochemical and genetic evidence supports that ceramide can inhibit PI 3-kinase and Akt, the downstream target of the PI 3-kinase (Zhou et al. 1998; Zundel and Giaccia 1998; Zundel et al. 2000). Interestingly, acid SMase was found to be critical for the ceramide-induced inhibition of PI 3-kinase in endothelial cells (Zundel et al. 2000). In response to irradiation, acid SMase-dependent ceramide generation induced the association of caveolin-1 with PI 3-kinase leading to inhibition of lipid kinase activity (Zundel et al. 2000). Since PC12 cells do not express significant amounts of caveolin-1 (Bilderback et al. 1999), our results suggest that the reciprocal interaction may also occur, at least in the absence of caveolin-1. Indeed, in MCF-7 human breast cancer cells that express low levels or no caveolin-1 (Engelman et al. 1999), PI 3-kinase also suppressed TNF-induced ceramide generation and apoptosis (Burow et al. 2000). The role of acid SMase in generating bioactive pools of ceramide appears to depend on the nature of the stimulus, the strength of the inducing signal as well as the cell type (Lin et al. 2000). Therefore, it will be important to determine if the presence of caveolin-1, or other caveolar proteins, may be critical in defining the cell specific role of acid SMase and PI 3-kinase in regulating ceramide-mediated stress responses.

In summary, we have demonstrated that PI 3-kinase can negatively regulate p75NTR-dependent sphingolipid signaling through the inhibition of acid SMase. These data raise the possibility that the PI 3-kinase/Akt pathway may serve as a convergence point for regulation of survival and stress signaling between Trk A tyrosine kinase and p75NTR-dependent sphingolipid signaling

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr Sung Ok Yoon for critical review of the manuscript and Dr R. Kolesnick for providing the FLAG-tagged acid SMase cDNA. This work was supported by grants NS38154 and NS38745 from the National Institutes of Health and a Career Development Award from the Juvenile Diabetes Foundation International to RTD. TRB is supported by a National Research Service Award.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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