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- Materials and methods
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.
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).
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 18.104.22.168)/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 22.214.171.124)/acid sphingomyelinase (acid SMase) which localizes to caveolae-related domains.
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- Materials and methods
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.
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