MOLECULAR AND CELLULAR BIOLOGY, Apr. 2001, p. 2695–2705 0270-7306/01/$04.00⫹0 DOI: 10.1128/MCB.21.8.2695–2705.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 21, No. 8
Sustained Signaling by Phospholipase C-␥ Mediates Nerve Growth Factor-Triggered Gene Expression DEOG-YOUNG CHOI,1† JUAN JOSE TOLEDO-ARAL,1‡ ROSALIND SEGAL,2 1 AND SIMON HALEGOUA * Department of Neurobiology & Behavior, State University of New York at Stony Brook, Stony Brook, New York 117945230,1 and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 021152 Received 16 November 2000/Returned for modification 21 December 2000/Accepted 24 January 2001
In contrast to conventional signaling by growth factors that requires their continual presence, a 1-min pulse of nerve growth factor (NGF) is sufficient to induce electrical excitability in PC12 cells due to induction of the peripheral nerve type 1 (PN1) sodium channel gene. We have investigated the mechanism for this triggered signaling pathway by NGF in PC12 cells. Mutation of TrkA at key autophosphorylation sites indicates an essential role for the phospholipase C-␥ (PLC-␥) binding site, but not the Shc binding site, for NGF-triggered induction of PN1. In concordance with results with Trk mutants, drug-mediated inhibition of PLC-␥ activity also blocks PN1 induction by NGF. Examination of the kinetics of TrkA autophosphorylation indicates that triggered signaling does not result from sustained activation and autophosphorylation of the TrkA receptor kinase, whose phosphorylation state declines rapidly after NGF removal. Rather, TrkA triggers an unexpectedly prolonged phosphorylation and activation of PLC-␥ signaling that is sustained for up to 2 h. Prevention of the elevation of intracellular Ca2ⴙ levels using BAPTA-AM results in a block of PN1 induction by NGF. Sustained signaling by PLC-␥ provides a means for differential neuronal gene induction after transient exposure to NGF.
for the acquisition of electrical excitability, was shown to be independent of key components of this pathway (14). Thus a brief, transient exposure to NGF can have long-lasting effects on electrical excitability, independent of its effects on other neuronal phenotypic traits or survival. A mechanistic analysis of signaling pathways resulting from transient NGF exposure is warranted to understand both the differential control of neuronal traits and the means by which NGF-induced signals may be rapidly reversible or prolonged after NGF withdrawal. The mechanism by which transient exposure to NGF can have selective, long-term consequences poses a formidable challenge for signal transduction. As is generally true for growth factor receptor tyrosine kinases, the established mechanism for Trk-mediated signaling involves Trk autophosphorylation, followed by the tyrosine phosphorylation and activation of substrate proteins which dock to the specific Trk autophosphorylation sites (see reference 20). In PC12 cells, Trk autophosphorylation mediates two main signaling pathways (45). The activation of Shc, which binds to the autophosphorylated site of TrkA at Tyr490, leads to stimulation of the proto-oncoprotein signaling pathway. Activation of this pathway is rapidly reversible due to deactivation of several intermediary components, for example, by the intrinsic GTPase activity of Ras itself (see reference 5) and by the action of specific MAP kinase phosphatases (see, e.g., reference 6). The reversibility of this pathway would explain the requirement for continual, long-term stimulation by NGF to elicit neurite growth and other downstream effects. Phosphorylation of the other major signaling site on TrkA, Tyr785, mediates docking and activation of Phospholipase C-␥ (PLC-␥). PLC-␥ signaling is of major importance in mediating growth factor actions both in vivo and in vitro, as is particularly apparent for the control
Nerve growth factor (NGF) stimulates expression of the peripheral nerve type 1 (PN1) voltage-dependent sodium channel gene (henceforth designated the PN1 gene or PN1) both in vitro (15) and in vivo (17), which in PC12 cells results in the acquisition of electrical excitability. The PN1 gene is induced by an unusual, heretofore unexplained mechanism whereby a transient, 1-min treatment with NGF results in longterm induction of the gene, referred to as a triggered pathway (48). Conventional signaling by NGF, as by other growth factors, requires continuous stimulation of its receptor to mediate long-term actions, such as the induction of neurite growth and other neuronal traits and the promotion of neuronal survival. The biological effects of NGF are mediated primarily through its receptor tyrosine kinase, TrkA (29, 30), which stimulates intracellular signaling pathways leading to specific protein phosphorylations (24) and differential gene inductions (see reference 23). TrkA engages the activation of a branched (14) proto-oncoprotein signaling pathway involving Shc (36, 40), Src (31), Ras (22), Raf (37, 50), mitogen-activated protein kinase (MAPK) (11, 16, 47), and other downstream effectors (20, 44). Despite the importance of this proto-oncoprotein signaling pathway for many of NGF’s major actions including neurite growth and gene inductions, stimulation of transcription from voltage-dependent sodium channel genes, required * Corresponding author. Mailing address: Department of Neurobiology & Behavior, State University of New York at Stony Brook, Stony Brook, NY 11794-5230. E-mail:
[email protected]. † Present address: Life Science Research Institute, LG Chem Research Park, Yousong-gu, Taejon 305-380, Korea. ‡ Present address: Departamento de Fisiologia Medica y Biofisica, Facultad de Medicina, Universitad de Sevilla, Sevilla 41009, Spain. 2695
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of mitogenesis and the regulation of gene expression in many nonneuronal cells (see reference 7). In mediating NGF actions, the long-term effects of the PLC-␥ signaling pathway have been largely unknown, although it has been suggested to provide a minor contribution to Ras signaling (45) and is required for the enhanced expression of the neuronal intermediate filament protein gene, peripherin (34). We have investigated the contributions of the major modes of NGF signaling to the triggered pathway that mediates PN1 gene induction in PC12 cells. This investigation has resulted in the identification of PLC-␥ in a specific Trk signaling pathway mediating PN1 gene induction. Kinetic analysis of PLC-␥ signaling revealed a remarkably triggered pathway, which mediates the acquisition of electrical excitability. MATERIALS AND METHODS Materials. Recombinant human NGF- (Genentech) and fibroblast growth factor 1 (FGF-1) (kindly provided by M. Jaye) were each used at concentrations of 50 ng/ml. 4-␣-Phorbol, phorbol-12-myristat-13-acetate (PMA), phorbol-12,13dibutyrate (PDBU), GF109203X, U73122, and U73343 (Calbiochem) were dissolved in dimethyl sulfoxide at a 1,000⫻ final concentration. Ionomycin (Calbiochem) was dissolved in methanol and used at a final concentration of 5 to 10 M. BAPTA-AM and its derivatives (Molecular Probes, Inc.) were dissolved in dimethyl sulfoxide at a 1,000⫻ final concentration. Anti-TrkA antibody and antiphospho-Tyr490 (P-Y490) antibody were generated as previously described (43). Anti-protein kinase C-ε antibody and anti-PLC-␥1 antibody were from Upstate Biotechnology, Inc. Anti-N-CAM monoclonal antibody was a kind gift from James Trimmer. Recombinant protein G-agarose was from GIBCO-BRL. Antirabbit or mouse immunoglobulin G donkey antibody conjugated to horseradish peroxidase was from Amersham. ECL kits were from New England Nuclear. RNasin, DNase I SP6 polymerase, T7 polymerase, and all riboprobe reagents were from Promega. The radioisotope [␣-32P]UTP (800 Ci/mmol) was from New England Nuclear. Anti-phospho-Tyr785 (P-Y785). The phospho-specific antibodies to the P-Y 785 site were generated using previously described methods (42). Briefly, rabbits were immunized with a phosphopeptide (CLHALGKATPI-P-Tyr-LD) corresponding to the carboxyterminus of TrkC. The immunoglobulin fraction was purified on a protein A-Sepharose column. The immunoglobulin was then applied to an Affigel column to which the unphosphorylated carboxy-terminal TrkC peptide had been attached. Following binding and washing of the column, the column was washed with 10 mM Tris and 500 mM NaCl, and then the phosphospecific antibody was eluted with 0.1 M glycine (pH 3.0). The affinity-purified antibody was tested with peptide competition. The phosphopeptide eliminated binding, but the unphosphorylated peptide did not. Phosphopeptides corresponding to the Y490 and Y674.5 sites also did not compete for binding. Cell culture. PC12 (21) and PC12-derived transfectants were grown on tissue culture dishes in Dulbecco’s modified Eagle’s medium (GIBCO-BRL) supplemented with 10% donor horse serum (JRH Biosciences), 5% fetal bovine serum (JRH Biosciences), and 1% penicillin-streptomycin (GIBCO-BRL) in an atmosphere of 10% CO2 at 37°C. Pulsatile treatment of cells with NGF was performed as described previously (48). Briefly, after treatment with NGF for 1 to 2 min at 37°C in growth medium, the medium was removed, and the cells were washed four times with warm (37°C) culture medium. Cells were then reincubated for the duration of the experiment. In all of the experiments, the control cells were similarly washed with fresh culture media at the time of experiment to control for serum-induced responses. Protein kinase C translocation. PC12 cells were washed two times with icecold phosphate-buffered saline (PBS), and the harvested cells were resuspended with lysis buffer (20 mM Tris [pH 7.6], 50 mM -mercaptoethanol, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF] followed by two 10-s bursts of sonication at 20 W with 10-s intervals between bursts. The lysates were separated into cytosolic and membrane fractions by centrifugation at 12,000 ⫻ g for 20 min at 4°C. Subsequently, membrane fractions (pellet) were extracted with lysis buffer also containing 1% NP-40 and 50 mM EGTA for 30 min at 4°C. Thereafter, the extracted proteins were recovered after centrifugation at 12,000 ⫻ g for 20 min at 4°C. The protein concentration was determined using Coomassie Plus protein assay reagent (Pierce). A total of 50 g of extracted proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electro-
MOL. CELL. BIOL. phoresis (SDS-PAGE) on a 7.5% polyacrylamide gel and further analyzed by Western blotting as described below. Western blot analysis. Cells were washed in PBS and then lysed in lysis buffer (20 mM Tris [pH 7.6], 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, and 1% NP-40). The lysates were clarified by centrifugation at 12,000 ⫻ g for 10 min at 4°C to remove nuclei. The protein concentration in the lysates was determined using Coomassie Plus protein assay reagent (Pierce). A total of 50 to 100 g of extracted proteins from each sample was subjected to SDS-PAGE on a 7.5% polyacrylamide gel. The electrophoresed proteins were transferred onto a nitrocellulose membrane using a Bio-Rad Trans Blot according to the manufacturer’s instructions. After incubation for 1 h with blocking solution (5% nonfat dry milk in PBS), blots were probed with primary antibody for 2 h at room temperature or overnight at 4°C. While all other antibodies are diluted in blocking solution, mouse anti-phosphotyrosine antibody (4G10, kindly provided by B. Druker) was diluted in PBS containing 3% bovine serum albumin, 10 mM Tris (pH 7.6), 1 mM Na3VO4, 1 mM EDTA, and 0.05% Tween 20. The blots were then probed with secondary anti-rabbit or anti-mouse immunoglobulin G donkey antibody conjugated with horseradish peroxidase (Amersham) for 1 h at room temperature. The blots were stained for 1 min using an ECL kit (Amersham) and exposed to X-ray film (Kodak). X-ray films were scanned with Scanmaster 3⫹, and the bands were quantitated with NIH Image 1.62b7 software. Immunoprecipitation. Cells were washed in PBS and then lysed with lysis buffer (20 mM Tris [pH 7.6], 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, and 1% NP-40). The lysates were clarified by centrifugation at 12,000 ⫻ g for 10 min at 4°C. The protein concentration of lysates was determined using Coomassie Plus protein assay reagent (Pierce). About 1 mg of cell lysates was diluted in lysis buffer and incubated at 4°C for 4 h with 5 g of primary antibody. Immune complexes were isolated using 50 l of protein Gagarose suspension (50% [vol/vol]) (GIBCO-BRL). The immunoprecipitates were washed three times with lysis buffer and then eluted from protein G-agarose beads by treatment with SDS-PAGE sampling buffer. After being placed in a boiling bath for 5 min, supernatants were subjected to SDS–7.5% PAGE and analyzed by Western blotting. Northern blot analysis. Total cellular RNA was isolated from cells, and Northern blot analysis was carried out as previously described (14). [␣-32P]UTPlabeled anti-sense RNA probes were synthesized using the following linearized cDNA clones as templates: RB211 (10), encoding a conserved sodium channel coding region and pIB15 (12), encoding cyclophilin, subcloned into pSP65; pG7TR1, encoding transin (35), subcloned into pGEM7Zf(⫹); pPeripherin, encoding a partial sequence of rat peripherin (bp 1159 to 1503) (32) which was isolated by PCR from a PC12 deoxyribosylthymine-random cDNA library, subcloned into a pBluescript II SK(⫹) plasmid; pST4, encoding Thy-1 (27), subcloned into pSP65. All riboprobes were generated according to the manufacturer’s instruction using SP6 polymerase (Promega) except for peripherin probes, which were synthesized using T7 polymerase. The blots were washed twice in 2⫻ SSC (1⫻ SSC is 0.15 M Nacl plus 0.015 M sodium citrate)–0.1% SDS at 68°C and twice in 0.2% SSC–0.1% SDS for 20 min at 68°C. PN1 mRNA bands were quantitated with a Phosphor Imager (Molecular Dynamics), and all values were normalized to the level of cyclophilin mRNA.
RESULTS To determine if induction of the PN1 gene by a brief pulse of NGF requires the NGF receptor tyrosine kinase, TrkA, PN1 gene expression was examined in PC12 mutant nnr5 cells, which lack Trk (18, 33). Neither continuous nor pulsatile 2-min) exposure to NGF was able to stimulate the induction of PN1 in these cells (Fig. 1A), indicating that PN1 induction cannot be stimulated by the p75 NGF receptor expressed in these cells. However, the response to NGF could be rescued by introduction of TrkA, as seen using nnr5-T14 cells, which are stable nnr5 transfectants expressing TrkA (Fig. 1A). To determine if Trk tyrosine kinase activity was required for this NGF action, PC12 cells were treated with the Trk kinase inhibitor, K252a, in addition to NGF. As shown in Fig. 1B, K252a was a potent inhibitor of PN1 induction at a concentration known to
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FIG. 1. TrkA tyrosine kinase activity is required for NGF induction of PN1. (A) Total cellular RNA (10 g) was isolated from PC12 nnr5 (lacking TrkA) and nnr5-TrkA (T14 cells) cells expressing wild-type TrkA, at 5 h after either a 2-min pulsatile treatment or continuous treatment with 50 ng of NGF per ml. RNA was electrophoresed through a 0.8% agarose gel and blotted. Blots were hybridized with a sodium channel probe (top) and rehybridized with a probe specific for the internal control cyclophilin (bottom). (B) PC12 cells were pretreated with 150 nM K252a for 20 min and then treated with 50 ng of NGF per ml for 5 h. Northern blots were prepared and analyzed as described in the legend to panel A.
specifically inhibit Trk kinase activity in PC12 and other cells (3, 46). Two autophosphorylation sites have been defined on TrkA which mediate docking and activation of Shc (Y490) and of PLC-␥ (Y785). To identify the signaling pathway involved in triggered PN1 induction, nnr5 cell transfectants expressing TrkA mutants, in which these sites have been mutated, were used (45). Each mutant is capable of TrkA kinase activation and autophosphorylation at multiple sites after NGF binding (reference 45 and Fig. 2A,) although the wild-type TrkA kinase shows a higher specific kinase activity (see Fig. 2A). Antiphosphotyrosine antibodies specific to each site were used to reveal the specific defects in autophosphorylation of each mutant. As reported previously, the anti-P-Y490 antibody was able to bind well to NGF-treated mutant Y785F but not to the NGF-treated Y490F mutant (reference 43 and Fig. 2B). To carry out a similar analysis of the Y785 phosphorylation, a phospho-specific anti-P-Y785 antibody was generated (see Materials and Methods). Anti-P-Y785 bound well to the Y490F mutant after NGF stimulation, but not to the Y785F mutant (Fig. 2B). As expected, both antibodies bound to NGF-treated wild-type TrkA, and neither of the antibodies bound to the Y490F-Y785F double mutant (data not shown). To verify that the TrkA mutant-expressing cells were defective in specific signaling pathways, the stimulated phosphorylation of Shc
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(p52Shc) (41) and PLC-␥ were examined by Western blotting (Fig. 2C and D). Anti-phosphotyrosine-specific antibody bound well to Shc after NGF treatment of nnr5 cells expressing wild-type and Y785F mutant TrkAs, but very poorly to Shc from TrkA Y490F-expressing cells. Similarly, anti-phosphotyrosine antibody bound well to PLC-␥ after NGF treatment of cells expressing wild-type and Y490F mutant TrkAs, but not to PLC-␥ from TrkA Y785F-expressing cells (reference 43 and Fig. 2C). These TrkA mutant-expressing cells were also characterized by examining NGF induction of the transin gene, which is induced by a Ras-dependent pathway (14), and of the peripherin gene, which is induced by PLC-␥ stimulation (34). As shown in Fig. 2E and F, NGF-induced expression of transin mRNA, but not peripherin mRNA, was defective in nnr5 cells expressing TrkA Y490F. In nnr5 cells expressing TrkA Y785F, the level of transin induction was similar to that seen with wild-type TrkA, but as previously reported (34), the induction of peripherin mRNA in these cells was defective (Fig. 2F). The ability of NGF treatment to induce PN1 mRNA was examined in the various nnr5 TrkA transfectants to determine if the Shc or PLC-␥ signaling pathways were involved in PN1 gene induction. As shown in the Northern blot in Fig. 3A, PN1 was induced normally in cells expressing either wild-type or Y490F mutant TrkAs, both by continuous and pulsatile (1min) NGF treatments. However, when examined in two different clonal isolates of nnr5 transfectants expressing the Y785F mutant TrkA, induction of PN1 mRNA was defective in response to pulsatile NGF treatment (Fig. 3A). The results of four independent experiments indicated that PN1 induction by TrkA Y785F is on average 78% (standard deviation ⫽ ⫾7%) lower than that seen in cells expressing either wild-type TrkA or the Y490F mutant. The same TrkA mutant was equally defective in mediating PN1 induction in response to a 5-h continual NGF treatment, suggesting that PN1 induction by NGF is mediated by a single Trk signaling event. PN1 induction was completely blocked (94% ⫾ 1%) in NGF-treated nnr5 transfectants expressing the double mutant TrkA Y490FY785F (see Fig. 3A). Longer NGF treatment times, from 5 to 24 h, failed to generate any significant accumulation of PN1 mRNA in any transfectant expressing a Y785F TrkA mutation (data not shown). To ensure that cells expressing the Y785F mutant TrkA were not generally defective in signaling PN1 gene induction, the nnr5 transfectants expressing wild-type TrkA or the TrkA mutants were treated with FGF-1. After 5 h of FGF treatment, PN1 mRNA was found to be well induced in the transfectant line expressing Y785F and also, although to a lesser extent, in cells expressing Y785F-Y490F (Fig. 3B). The dependence of PN1 gene induction on the PLC-␥ binding site of Trk at Y785 suggests that PLC-␥ activity may be necessary to mediate PN1 induction by NGF. To test for this possible requirement of PLC-␥, the inhibitor U73122 was used. PC12 cells were treated with NGF in the presence or absence of either the active PLC-␥ inhibitor U73122 or the inactive derivative U73343, and PN1 mRNA was assessed after 5 h. As shown in Fig. 3C, PN1 mRNA induction by NGF was blocked by over 80% in cells treated with the PLC-␥ inhibitor U73122 but unaffected by a similar treatment with U73343. The above results indicated that the autophosphorylation of TrkA at Y785 mediates PLC-␥ signaling of the triggered induction of PN1 by NGF. Because the induction of transin
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FIG. 2. Specific signaling defects of TrkA mutants. (A) PC12 nnr5 cells expressing wild-type TrkA, TrkA Y490F, and TrkA Y785F were treated with 50 ng of NGF per ml for 5 min. Cell lysates were electrophoresed through a SDS–7.5% polyacrylamide gel and blotted onto a nitrocellulose filter. The blot was probed with anti-TrkA antibody (top) and an anti-phosphotyrosine (4G10) antibody (bottom). (B) PC12 nnr5 Y490F and Y785F cells were treated with 50 ng of NGF per ml for 5 min. Cell lysates were electrophoresed and blotted onto nitrocellulose. The blots were probed with antibodies specific for individual phosphotyrosine residues of TrkA (anti-P-Y490 or anti-PY785). (C) PC12 nnr5 wild-type (WT), Y490F, and Y785F cells were treated with 50 ng of NGF per ml for 5 min. Whole-cell lysates were electrophoresed and blotted, and the blots were probed with antiphosphotyrosine antibody. (D) PC12 nnr5 WT, Y490F, and Y785F cells were treated with 50 ng of NGF per ml for 5 min. PLC-␥ was immunoprecipitated from cell lysates using anti-PLC-␥ antibody. Immunoprecipitates were electrophoresed and blotted onto nitrocellulose. The blots were probed with anti-phosphotyrosine antibody (top). The blots were then stripped and reprobed with anti-PLC-␥ antibody (bottom). (E) PC12 nnr5 WT, Y490F, and Y785F cells were treated with 50 ng of NGF per ml for 5 h. Total cellular RNA (10 g) was isolated and electrophoresed through 0.8% agarose gels and blotted. Blots were hybridized with a probe specific for transin (1.9 kb) (top). The blots were rehybridized with a probe specific for cyclophilin (1 kb) as an internal standard (bottom). (F) Cells were treated with 50 ng of NGF per ml for 48 h. Total cellular RNA (10 g) was isolated and electrophoresed through 1.4% agarose gels and blotted. The blot was hybridized with a probe specific for peripherin (top) or with a probe specific for the internal control cyclophilin (bottom).
mediated by autophosphorylation of TrkA at Y490 is not triggered, we examined the kinetics of TrkA autophosphorylation at these two sites to determine if triggering might be explained by a differential time course of phosphorylation. To facilitate the analysis of TrkA autophosphorylation using site-specific anti-phospho-specific antibodies, Trk-PC12 cells which overexpress TrkA were used, as was done previously (43). Trk-PC12 cells were treated with NGF either continuously for times up to 30 min or for 2 min followed by a chase period in the absence of NGF for up to 30 min. The phospho-specific anti-P-Y490 or anti-P-Y785 antibodies were exploited to monitor the phosphorylation state of TrkA at Y490 and Y785 with Western blot analysis. To facilitate this analysis, the TrkA-PC12 cell trans-
fectant, which overexpresses TrkA (26), was used. As shown in Fig. 4, autophosphorylation of TrkA at each site was maintained throughout the 30 min of continual NGF treatment. However, after a 2-min pulse treatment with NGF, the extent of phosphorylation at each of the sites rapidly diminished to near baseline within the 30-min chase period. No differences were seen between the rates of decay of phosphorylation at each of the sites. Similar results were obtained using PC12 cells from which TrkA was immunoprecipitated (data not shown). These results suggest that the triggered signal is not mediated at the level of TrkA kinase activity, autophosphorylation, or dephosphorylation. A comparison of signaling pathway kinetics was continued
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FIG. 3. NGF-induced PN1 expression is specifically blocked in cells expressing TrkA Y785F. (A) Total cellular RNA (10 g) was isolated from PC12 nnr5 wild-type (WT), Y490F, Y785F, and Trk Y490F/785F cells at 5 h after either a 1-min pulsatile or continuous treatment with 50 ng of NGF per ml. RNA was electrophoresed and blotted, and the blots were probed with a sodium channel probe, pRB211, and rehybridized with a cyclophilin probe, p1B15. (B) Total cellular RNA (10 g) was isolated from PC12 nnr5 WT, Y490F, Y785F, and Trk Y490F-785F cells at 5 h after treatment with 50 ng of FGF-1 per ml. RNA was hybridized with a sodium channel probe, pRB211 (top), and rehybridized with a probe specific for cyclophilin (bottom). (C) Northern blots prepared from total cellular RNA (10 g) isolated from PC12 cells at 5 h after treatment with indicated amounts of U73343, U73122, and/or 50 ng of NGF per ml (cells were also pretreated with the inhibitors for 30 min before NGF treatment) were hybridized with a sodium channel probe (top) and rehybridized with a cyclophilin probe for an internal control (bottom).
with the downstream effectors, Shc and PLC-␥, which are each phosphorylated on tyrosine after binding to the TrkA P-Y490 and P-Y785 autophosphorylation sites, respectively. Again, cells were treated with NGF either continuously for up to 60 min or pulsed with NGF for 2 min and then chased without NGF for up to 60 min. The levels of Shc and PLC-␥ phosphorylation were examined by probing Western blots of these proteins with anti-P-Tyr antibody. As shown in Fig. 5, and unlike the case with the different autophosphorylation sites on TrkA, the kinetics of tyrosine phosphorylation of these effector proteins were quite different from each other. As expected, the tyrosine phosphorylation of Shc followed a pattern of phosphorylation similar to that of the TrkA PY-490 tyrosine auto-
phosphorylation site to which it binds. After a 2-min pulse with NGF, Shc phosphorylation was stimulated within 5 min but diminished rapidly within the 30-min chase period. As previously reported (45), Shc phosphorylation remained stimulated during continual NGF stimulation, but at a level which decreased with time (see Fig. 5A and C). In contrast, the time course of PLC-␥ phosphorylation was markedly prolonged. After a pulse of NGF treatment, PLC-␥ became phosphorylated at a significantly higher level (about 40% higher) than with continual NGF treatment. The stimulated level of PLC-␥ tyrosine phosphorylations was comparably maintained whether the cells were continuously treated or pulsed with NGF followed by a 1-h chase period (Fig. 5B and C). As a result, 1 h
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FIG. 4. Both Y785 and Y490 sites are transiently phosphorylated after pulsatile NGF treatment. Protein extracts were prepared from TrkA PC12 cells at the indicated times after either a 2-min pulsatile (pulse-chase) treatment or continuous treatment with 50 ng of NGF per ml. The proteins were electrophoresed through a SDS–7.5% polyacrylamide gel and blotted onto nitrocellulose. The blot was probed with anti-P-Y490 antibody (top). A parallel blot made from the same samples was probed with anti-P-Y785 antibody (middle). After stripping, the blots were reprobed with anti-TrkA antibody (bottom).
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after initial exposure to NGF, the level of PLC phosphorylation after transient exposure was higher (75% of control) than with continual exposure (60% of control) (Fig. 5C). In contrast, the level of Shc at 1 h after transient exposure was less than 35% of maximal compared to 60% of maximal with continual exposure (Fig. 5C). These differences between the magnitude and time course of Shc and PLC-␥ phosphorylation were reproducibly seen with quantitatively similar results. The results with PLC-␥ and Shc phosphorylation suggested that PLC-␥ activation remained high for at least 60 min after only a pulse treatment of cells with NGF, while the activation of Shc was much more transient. If these differences reflected their signaling potentials after pulsed NGF treatment, then activation of PLC-␥ and Shc downstream effectors would similarly exhibit different kinetics. To test this possibility, we compared the kinetics of activation of the Shc and PLC-␥ down-
FIG. 5. Pulsatile NGF treatment triggers prolonged tyrosine phosphorylation of PLC-␥, but not of Shc. (A) Cell lysates were prepared from PC12 cells at the indicated times after either 2 min of pulsatile (pulse-chase) treatment or continuous treatment with 50 ng of NGF per ml. The proteins were electrophoresed and blotted onto nitrocellulose, and the blots were probed with anti-phosphotyrosine antibody (anti-P-Tyr) (top). After being stripped, the blot was probed again with anti-Shc antibody (bottom). (B) PLC-␥ was immunoprecipitated from the cell lysates with anti-PLC-␥ antibody. Western blots of the imunoprecipitated proteins were probed with anti-phosphotyrosine antibody (top). After being stripped, the blot was probed with anti-PLC-␥ antibody (bottom). (C) Quantification was performed using a densitometer and software as described in Materials and Methods. The maximal response to continuous NGF treatment was given a value of 1.0.
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FIG. 6. Pulsatile NGF treatment triggers prolonged activation of protein kinase C, but not of MAPK. (A) Cell lysates were prepared from PC12 cells at the indicated times after either 2 min of pulsatile (pulse-chase) treatment or continuous treatment with 50 ng of NGF per ml. The proteins were electrophoresed and blotted onto nitrocellulose, and the blots were probed with anti-phosphotyrosine antibody. The positions of the ERK1 and ERK2 MAPKs are indicated. (B) At the indicated times after either 2 min of pulsatile (pulse-chase) treatment or continuous treatment with 50 ng of NGF per ml, PC12 cells were fractionated into cytosolic and membrane fractions by sonication and subsequent centrifugation. Membrane proteins were extracted, electrophoresed, and blotted, and the blots were probed with anti-protein kinase C-ε (anti-PKC ε) antibody (top) and with anti-N-CAM antibody (bottom). (C) Quantification was performed as described in Materials and Methods. The maximal response to continuous NGF treatment was given a value of 1.0.
stream signaling effectors MAPK and protein kinase C, respectively, after pulse treatment with NGF. Each of these effectors has been reported to be activated with continuous NGF treatment of PC12 cells (39, 51). To examine MAPK, long-term tyrosine phosphorylation on Western blots of MAPK probed with anti-P-Tyr antibody was monitored. As shown in Fig. 6A and C, the tyrosine phosphorylation of both the extracellular signal-regulated kinase 1, (ERK1) and ERK2 MAPKs is quite transient after a 2-min pulse of NGF treatment, decreasing by 70% within 15 min after NGF removal. The activation of protein kinase C-ε was monitored by the translocation of the kinase to the membrane fraction, which has been shown to persist with continual NGF treatment of the cells (51). As was seen with PLC-␥ activation, protein kinase C-ε was stimulated to a higher level after a 2-min pulse of NGF than with continuous treatment, and the stimulation persisted to a similar extent after pulse and continuous NGF treatments (Fig. 6B and C).
Because the PLC-␥ binding site on TrkA is required for triggered PN1 induction by NGF, we tested whether we could mimic PN1 induction by a selective stimulation of downstream signaling components. PLC-␥ activation stimulates two main signaling effectors, protein kinase C and elevated intracellular Ca2⫹ levels (via the action of IP3). To examine the ability of protein kinase C to mediate PN1 gene induction, cells were treated with phorbol ester derivatives, which are potent activators of protein kinase C. As shown by the Northern blot in Fig. 7A, treatment of cells with one of the protein kinase C activators, PMA or PDBU resulted in induction of PN1 mRNA in a time course similar to that seen with NGF treatment. Treatment of cells with the inactive derivative, 4-␣-phorbol, had no effect. Similarly, when cells were treated with the calcium ionophore, ionomycin, to raise intracellular Ca2⫹ levels, induction of PN1 mRNA was also observed (Fig. 7B). In each case, the level of PN1 induction was lower (about half) than that seen with NGF treatment. These results suggest that
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NGF treatment (Fig. 8C). The ability to inhibit NGF-mediated PN1 induction was compared among several BAPTA-AM derivatives with differing affinities for Ca2⫹. As expected, BAPTA-AM and dimethyl BAPTA-AM, which have high affinity for Ca2⫹ (in the nanomolar range), effectively inhibited NGF-mediated PN1 induction (90% inhibition), while tetrafluoro-BAPTA-AM, which has a low affinity for Ca2⫹ (in the micromolar range) was poorly effective (20% inhibition) in inhibiting PN1 induction (Fig. 8D). To ensure that BAPTA-AM treatment of cells did not nonspecifically block the expression of PN1 mRNA, its effect on PMA-induced PN1 mRNA was examined. BAPTA-AM treatment had no effect on the ability of PMA to induce PN1 mRNA (Fig. 8E). DISCUSSION
FIG. 7. PN1 mRNA expression is induced by both protein kinase C activators and calcium ionophore. (A) Northern blots prepared from total cellular RNA (10 g) isolated from PC12 cells at 5 h after treatment with 0.1 M 4-␣-phorbol, PMA, PDBU or 50 ng of NGF per ml were hybridized with a sodium channel probe (top) and rehybridized with cyclophilin probe for an internal control (bottom). (B) Northern blots prepared from total cellular RNA (10 g) isolated from PC12 cells at 5 h after treatment with PMA, ionomycin, or 50 ng of NGF per ml were hybridized with a sodium channel probe (top) and rehybridized with cyclophilin probe for an internal control (bottom).
stimulation of either arm of the PLC-␥ signaling pathway is capable of stimulating the induction of PN1 mRNA. To determine whether the PLC-␥ signaling effectors were required for the induction of PN1 by NGF, we examined the effects of blocking protein kinase C or blocking the elevation of intracellular calcium levels on PN1 induction. Treatment of cells with the inhibitor of protein kinase C, GF109203X (49), resulted in a complete inhibition of PMA-stimulated PN1 or transin mRNA inductions (Fig. 8A). However, this inhibitor had little effect on the ability of NGF to mediate induction of either PN1 or transin (Fig. 8A). To prevent the elevation of intracellular Ca2⫹ levels by NGF, cells were loaded with the calcium chelator BAPTA, using the BAPTA-AM ester. Cells were treated or not with BAPTA-AM for 1 h before and during NGF treatment, and the levels of PN1 mRNA were examined by Northern blot analysis. As shown in Fig. 8B, BAPTA-AM mediated a dose-dependent inhibition of PN1 induction by NGF. At 50 M BAPTA-AM, a concentration at which it blocks intracellular calcium mobilization, a nearly complete inhibition of NGF-mediated PN1 induction was attained. However, BAPTA-AM did not inhibit the ability of NGF to induce transin mRNA levels, which occurs through the Ras signaling pathway, and instead resulted in an enhancement of this NGF effect (Fig. 8B and C). This enhancement of transin expression is consistent with the reported enhancement of MAP kinase signaling by growth factors, seen in the absence of intracellular Ca2⫹ (28). BAPTA-AM was equally effective at inhibiting PN1 induction mediated by pulsatile NGF treatment (which selectively induces PN1 over transin) as by continuous
The following conclusions may be drawn from the results presented: (i) the triggered induction of PN1 by NGF is a direct result of autophosphorylation of TrkA on Y785, which mediates the binding and activation of PLC-␥; (ii) the phosphorylation of PLC-␥ is rapid and is sustained for at least 1 h after TrkA kinase activation is terminated; (3) unlike the Shc 3 MAPK pathway, activation of PLC-␥ pathway downstream effectors is also sustained after brief activation of TrkA; and (iv) intracellular Ca2⫹ elevation is the predominant mediator of PN1 gene induction by NGF. Continual signaling by NGF, over a period of hours and days, is required for the expression of many neural-specific genes and for neurite growth. The expression of some of these genes, as exemplified by the vgf gene, requires persistent activation of the Ras signaling pathway, involving downstream mediators such as the transcriptional activator CREB and the immediate-early gene product NGFI-A (4, 13, 25). In contrast to most phenotypic traits, the early appearance of electrical excitability is due to a triggered Ras-independent mechanism, via induction of the voltage-dependent sodium channel gene, PN1 (14, 15). This triggered gene induction results in a peak time of PN1 mRNA expression at 5 h, following a pulse of NGF for as short as 1 min (48). The induction of PN1 mRNA at 5 h requires the prior expression of an unidentified immediate-early gene (48). Therefore, the signaling pathway to PN1 induction must encompass this immediate-early gene induction event. Mutational analysis of TrkA and the use of the PLC-␥ inhibitor U73122 demonstrated that PN1 induction depends on Y785, through which binding and activation of PLC-␥ occurs. PN1 induction was unaffected by the mutation Y490F, a site which mediates Ras signaling, consistent with previous results which indicated that Ras activation is neither necessary nor sufficient for PN1 induction (14). Although a small degree of Ras activation may occur through Y785 and PLC-␥ signaling (45), the mutation of Y490 severely blunted the Ras-dependent induction of the transin gene, without having a significant effect on the triggered induction of PN1. While these data indicate that PLC-␥ is the major pathway for PN1 induction, activation of Ras may also contribute to PN1 induction, as the level of induction of PN1 is lower in cells expressing a dominant-negative form of Ras (14), and PN1 induction is partially inhibited (by 30%) by the MEK inhibitor PD98059 (D.-Y. Choi and S. Halegoua, unpublished). Furthermore, PN1 induc-
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FIG. 8. Effects of the protein kinase C-specific inhibitor, GF109203X, and the intracellular calcium chelator, BAPTA-AM, on PN1 gene expression mediated by PMA and NGF. (A) PC12 cells were pretreated with 1 M GF109203X for 1 h and then treated with 50 ng of NGF per ml or 0.1 M PMA for 5 h. Northern blots of total cellular RNA (10 g) were probed with a sodium channel probe (top) and rehybridized with a probe specific for transin (middle) and a probe specific for cyclophilin (bottom). (B) PC12 cells were pretreated with 25 or 50 M BAPTA-AM for 1 h and then treated for 5 h with 50 ng of NGF per ml. Northern blots of total cellular RNA (10 g) were probed with a sodium channel probe pRB211 (top) and rehybridized with a probe specific for transin (middle) and a probe specific for cyclophilin (bottom). (C) PC12 cells were pretreated with 50 M BAPTA-AM for 1 h and then treated for 5 h with 50 ng of NGF per ml or for 2 min with NGF and incubated for a total of 5 h. Northern blots of total cellular RNA (10 g) were probed with a sodium channel probe (top) and rehybridized with a probe specific for transin (middle) and a probe specific for cyclophilin (bottom). (D) PC12 cells were pretreated with 40 M BAPTA-AM, dimethyl(DM) BAPTA-AM, or tetraflouro(TF)-BAPTA-AM for 1 h and then treated for 5 h with 50 ng of NGF per ml. Northern blots of total cellular RNA (10 g) were probed with a sodium channel probe (top) and rehybridized with a probe specific for cyclophilin (bottom). (E) PC12 cells were pretreated with 40 M BAPTA-AM for 1 h and then treated for 5 h with 50 ng of NGF per ml or 0.1 M PMA. Northern blots of total cellular RNA (10 g) were probed with a sodium channel probe (top) and rehybridized with a probe specific for cyclophilin (bottom).
tion is more severely defective in cells expressing the Trk double mutant Y490F-Y785F than in cells expressing TrkA Y785F (Fig. 3). Selective activation of the PLC-␥ downstream effectors protein kinase C and the elevation of intracellular Ca2⫹ were each able to mimic PN1 induction by NGF, and the chelation of intracellular Ca2⫹ prevented NGF from inducing the PN1 gene. Calcium elevation would thus appear to provide the predominant pathway by which NGF mediates triggered induction of the PN1 gene. Furthermore, the PLC-␥ pathway is required for any and all modes of PN1 mRNA induction seen
after 5 h of NGF treatment, because inhibition of this pathway blocks PN1 induction by both continual and pulsatile treatment with NGF. These results favor a single mechanism for the initial induction of PN1 expression. However, we cannot rule out the possible involvement of another unidentified signaling protein that may bind to the same site at Trk Y785. Kinetic analysis of Trk signaling and the signaling of effector pathways yielded insights into the mechanism by which PLC␥-mediated signaling is able to mediate the triggered gene induction event. This analysis excluded the possibility that differential phosphorylation or dephosphorylation of individual
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sites on Trk receptors could be responsible for triggered signaling, as the kinetics of phosphorylation at sites Y785 and Y490 were similar in response to a pulse of NGF treatment. Importantly, however, following a pulse of Trk activity, major differences were seen between the duration of the PLC-␥ and Shc phosphorylation states. The phosphorylation of Shc was transient, while PLC-␥ phosphorylation was hyperstimulated and persisted for at least 1 h after the short pulse of NGF. The prolonged time of PLC-␥ phosphorylation was reflected also in the apparent activation of protein kinase C, which was also hyperstimulated and remained membrane translocated for at least 1 h after a 2-min NGF pulse. On the other hand, both Shc phosphorylation and, more impressively, the phosphorylation of MAPK were much more transient, decreasing between 5 and 15 min after pulsatile NGF treatment. The rapid reversibility of the Shc3Ras3MAPK pathway is well documented, involving not only the dephosphorylation of the Shc shown here, but additionally at least the intrinsic GTPase activity of Ras and the activity of specific MAPK phosphatases. In contrast, our results indicate that the NGF-stimulated PLC-␥ signaling pathway is designed for long-term, triggered activation. The slow reversibility of PLC-␥ phosphorylation and activation may be explained most simply by the absence or inaccessibility of an appreciable level of PLC-␥ phosphatase activity toward its activated substrate after NGF treatment. Receptor-mediated long-term activation of PLC would not be expected to occur for other forms such as PLC-, whose activation kinetics are dictated by the timing of direct G protein activation, which is generally quite transient after receptor disengagement (see, e.g., reference 9). The prolonged activation of PLC-␥ would be expected to similarly stimulate phosphatidylinositol turnover, resulting in the long-term production of diacylglycerol and IP3. This is supported by the long-term stimulation of diacylglycerol-activated protein kinase C reported here. NGF treatment of PC12 cells has been shown to also result in an unusually slow and small, but persistent, elevation of intracellular Ca2⫹ resulting from stimulated phosphatidylinositol turnover (8, 38). Our results suggest that the long-lasting Ca2⫹ elevation may provide a necessary and sufficient signal for the triggered induction of the PN1 gene. Elucidating the mechanism by which this mode of Ca2⫹ signaling mediates gene induction will require identification of the Ca2⫹ activated transcription factors and their gene targets. How long must triggered signaling of PLC-␥ and its effectors persist after pulsatile NGF treatment in order to mediate PN1 gene induction? As shown previously, PN1 induction requires the prior induction of an immediate-early gene intermediate, expressed during the first 1.5 h after pulsatile NGF treatment (48). The PLC-␥ pathway remains activated for at least 1 h but not more than 2 h after pulsatile NGF treatment. This would certainly be long enough to mediate full expression of an immediate-early gene, as immediate-early genes such as c-fos can be maximally expressed within 30 min after the inducing stimulus (19). It would also suffice to mediate any post-translational activation of immediate-early gene products which may be required for their subsequent role in transcriptional transactivation. It would not, however, be long enough to directly mediate PN1 gene induction, since the induced PN1 mRNA is first seen only after 2 to 3 h of NGF treatment. PN1 gene induction thus could be explained by the time course of PLC-␥
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signaling only if the immediate early gene product is sufficient for PN1 induction. Our results lead to a model in which transient activation of TrkA by NGF results in the long-term activation of PLC-␥ and its effectors, such as IP3. The resulting prolonged elevation of intracellular Ca2⫹ mediates the induced expression of an immediate-early gene product(s), which then acts as a transcriptional transactivator, sufficient for the transcriptional activation of the PN1 gene. Although activation of TrkA at the cell surface may be sufficient to mediate the response, this model does not preclude a possible involvement of endocytosed TrkA, which could occur within the short time of pulsatile NGF treatment. This model could easily be extended to other neurotrophins and Trk receptors, as well as other growth factor receptor tyrosine kinases, which have similar signaling features (2). In this model, because the Shc-Ras-MAPK pathway is rapidly reversible, the short time of receptor tyrosine kinase activation would result in selective, prolonged activation of the PLC-␥ signaling pathway. As a result, transient exposure to growth factors would mediate differential signaling, resulting in the differential control of downstream events. In the case of NGF, the result is a selective control of electrical excitability and Na channel gene expression, as compared to neurite growth and the expression of genes giving rise to other neuronal phenotypic traits. It is likely that this PLC-␥ pathway is not specific for inducing the PN1 gene. Indeed, another gene which is induced through this pathway is that encoding peripherin, the neural-specific intermediate filament (34). Although peripherin gene induction occurs over a much longer period than PN1 (days versus hours), we have observed triggered induction of peripherin in PC12 cells, albeit to a lesser degree, 24 to 48 h after an NGF pulse (Choi and Halegoua, unpublished). The full array of phenotypic traits regulated in a triggered manner through the PLC-␥ signaling pathway remains to be determined. Triggered signaling by neuronal growth factor receptors, via prolonged activation of PLC-␥, provides a mechanism by which the selective control of a subset of biological effects is exerted by transient growth factor signaling in vivo. A single dose of exogenously administered NGF has been shown to result in rapid and long-term hyperalgesia, which includes the induction of PN1 (17). Normally, pulsatile delivery of neuronal growth factors in vivo could occur both during development and in the adult by synaptic release (1). Transient growth factor signaling in neurons would result in the selective, prolonged regulation of excitability, synaptic modulation, and other neuronal traits via changes in gene expression and in protein function mediated through the PLC-␥ pathway. The specificity in biological responses to neuronal growth factors may thus depend on both the kinetics of release and accessibility to the target. The complete array of paradigms that involve triggered signaling by growth factors in vivo may be of interest to elucidate. ACKNOWLEDGMENTS We thank L. Greene for providing the nnr cell line and all derived Trk transfectants, G. Valdez for constructing the pPeripherin clone, and G. Mandel for helpful comments and critical reading of the manuscript. This work was supported by grants from the National Institutes of Health, NS18218 to S.H. and NS 35148 to R.S.
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1. Altar, C. A., and P. S. DiStefano. 1998. Neurotrophin trafficking by anterograde transport. Trends Neurosci. 21:433–437. 2. Barbacid, M. 1995. Neurotrophic factors and their receptors. Curr. Opin. Cell Biol. 7:148–155. 3. Berg, M. M., D. W. Sternberg, L. F. Parada, and M. V. Chao. 1992. K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J. Biol. Chem. 267:13–16. 4. Bonni, A., D. D. Ginty, H. Dudek, and M. E. Greenberg. 1995. Serine 133-phosphorylated CREB induces transcription via a cooperative mechanism that may confer specificity to neurotrophin signals. Mol. Cell. Neurosci. 6:168–183. 5. Bourne, H. R., D. A. Sanders, and F. McCormick. 1990. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348:125–132. 6. Camps, M., A. Nichols, C. Gillieron, B. Antonsson, M. Muda, C. Chabert, U. Boschert, and S. Arkinstall. 1998. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280:1262–1265. 7. Carpenter, G., and Q. Ji. 1999. Phospholipase C-gamma as a signal-transducing element. Exp. Cell Res. 253:15–24. 8. Contreras, M. L., and G. Guroff. 1987. Calcium-dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC12 cells. J. Neurochem. 48:1466–1472. 9. Cook, B., M. Bar-Yaacov, H. Cohen Ben-Ami, R. E. Goldstein, Z. Paroush, Z. Selinger, and B. Minke. 2000. Phospholipase C and termination of Gprotein-mediated signalling in vivo. Nat. Cell Biol. 2:296–301. 10. Cooperman, S. S., S. A. Grubman, R. L. Barchi, R. H. Goodman, and G. Mandel. 1987. Modulation of sodium-channel mRNA levels in rat skeletal muscle. Proc. Natl. Acad. Sci. USA 84:8721–8725. 11. Cowley, S., H. Paterson, P. Kemp, and C. J. Marshall. 1994. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77:841–852. 12. Danielson, P. E., S. Forss-Petter, M. A. Brow, L. Calavetta, J. Douglass, R. J. Milner, and J. G. Sutcliffe. 1988. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261–267. 13. D’Arcangelo, G., R. Habas, S. Wang, S. Halegoua, and S. R. Salton. 1996. Activation of codependent transcription factors is required for transcriptional induction of the vgf gene by nerve growth factor and Ras. Mol. Cell. Biol. 16:4621–4631. 14. D’Arcangelo, G., and S. Halegoua. 1993. A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Raf-mediated gene inductions. Mol. Cell. Biol. 13:3146–3155. 15. D’Arcangelo, G., K. Paradiso, D. Shepherd, P. Brehm, S. Halegoua, and G. Mandel. 1993. Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways. J. Cell Biol. 122:915–921. 16. Gomez, N., and P. Cohen. 1991. Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature 353:170–173. 17. Gould, H. J., III, T. N. Gould, J. D. England, D. Paul, Z. P. Liu, and S. R. Levinson. 2000. A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain. Brain Res. 854:19–29. 18. Green, S. H., R. E. Rydel, J. L. Connolly, and L. A. Greene. 1986. PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. J. Cell Biol. 102:830– 843. 19. Greenberg, M. E., L. A. Greene, and E. B. Ziff. 1985. Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells. J. Biol. Chem. 260:14101–14110. 20. Greene, L. A., and D. R. Kaplan. 1995. Early events in neurotrophin signalling via Trk and p75 receptors. Curr. Opin. Neurobiol. 5:579–587. 21. Greene, L. A., and A. S. Tischler. 1976. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73:2424–2428. 22. Hagag, N., S. Halegoua, and M. Viola. 1986. Inhibition of growth factorinduced differentiation of PC12 cells by microinjection of antibody to ras p21. Nature 319:680–682. 23. Halegoua, S., R. C. Armstrong, and N. E. Kremer. 1991. Dissecting the mode of action of a neuronal growth factor. Curr. Top. Microbiol. Immunol. 165:119–170. 24. Halegoua, S., and J. Patrick. 1980. Nerve growth factor mediates phosphorylation of specific proteins. Cell 22:571–581. 25. Hawley, R. J., R. J. Scheibe, and J. A. Wagner. 1992. NGF induces the expression of the VGF gene through a cAMP response element. J. Neurosci. 12:2573–2581. 26. Hempstead, B. L., S. J. Rabin, L. Kaplan, S. Reid, L. F. Parada, and D. R. Kaplan. 1992. Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation. Neuron 9:883–896. 27. Ingraham, H. A., G. M. Lawless, and G. A. Evans. 1986. The mouse Thy-1.2 glycoprotein gene: complete sequence and identification of an unusual promoter. J. Immunol. 136:1482–1489.
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28. Ji, Q. S., and G. Carpenter. 2000. Role of basal calcium in the EGF activation of MAP kinases. Oncogene 19:1853–1856. 29. Kaplan, D. R., B. L. Hempstead, D. Martin-Zanca, M. V. Chao, and L. F. Parada. 1991. The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 252:554–558. 30. Klein, R., S. Q. Jing, V. Nanduri, E. O’Rourke, and M. Barbacid. 1991. The trk proto-oncogene encodes a receptor for nerve growth factor. Cell 65:189– 197. 31. Kremer, N. E., G. D’Arcangelo, S. M. Thomas, M. DeMarco, J. S. Brugge, and S. Halegoua. 1991. Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions. J. Cell Biol. 115:809–819. 32. Landon, F., M. Lemonnier, R. Benarous, C. Huc, M. Fiszman, F. Gros, and M. M. Portier. 1989. Multiple mRNAs encode peripherin, a neuronal intermediate filament protein. EMBO J. 8:1719–1726. 33. Loeb, D. M., J. Maragos, D. Martin-Zanca, M. V. Chao, L. F. Parada, and L. A. Greene. 1991. The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines. Cell 66:961–966. 34. Loeb, D. M., R. M. Stephens, T. Copeland, D. R. Kaplan, and L. A. Greene. 1994. A Trk nerve growth factor (NGF) receptor point mutation affecting interaction with phospholipase C-gamma 1 abolishes NGF-promoted peripherin induction but not neurite outgrowth. J. Biol. Chem. 269:8901–8910. 35. Matrisian, L. M., N. Glaichenhaus, M. C. Gesnel, and R. Breathnach. 1985. Epidermal growth factor and oncogenes induce transcription of the same cellular mRNA in rat fibroblasts. EMBO J. 4:1435–1440. 36. Obermeier, A., R. Lammers, K. H. Wiesmuller, G. Jung, 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. 268:22963–22966. 37. Oshima, M., G. Sithanandam, U. R. Rapp, and G. Guroff. 1991. The phosphorylation and activation of B-raf in PC12 cells stimulated by nerve growth factor. J. Biol. Chem. 266:23753–23760. 38. 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. 208:48–51. 39. Qui, M. S., and S. H. Green. 1992. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron 9:705–717. 40. Rozakis-Adcock, M., J. McGlade, G. Mbamalu, G. Pelicci, R. Daly, W. Li, A. Batzer, S. Thomas, J. Brugge, P. G. Pelicci, et al. 1992. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature 360:689–692. 41. Rusanescu, G., H. Qi, S. M. Thomas, J. S. Brugge, and S. Halegoua. 1995. Calcium influx induces neurite growth through a Src-Ras signaling cassette. Neuron 15:1415–1425. 42. Segal, R. A. 1997. Generation and utilization of phosphorylation-state specific antibodies to investigate neuronal signaling pathways. In C. J. Crawley, R. M. Gerfen, M. Rogawski, D. Sibley, P. Skolnick (ed.), Current protocols in neuroscience. Wiley, New York, N.Y. 43. Segal, R. A., A. Bhattacharyya, L. A. Rua, J. A. Alberta, R. M. Stephens, D. R. Kaplan, and C. D. Stiles. 1996. Differential utilization of Trk autophosphorylation sites. J. Biol. Chem. 271:20175–20181. 44. Segal, R. A., and M. E. Greenberg. 1996. Intracellular signaling pathways activated by neurotrophic factors. Annu. Rev. Neurosci. 19:463–489. 45. 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 12:691–705. 46. Tapley, P., F. Lamballe, and M. Barbacid. 1992. K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene 7:371–381. 47. Thomas, S. M., M. DeMarco, G. D’Arcangelo, S. Halegoua, and J. S. Brugge. 1992. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell 68:1031–1040. 48. Toledo-Aral, J. J., P. Brehm, S. Halegoua, and G. Mandel. 1995. A single pulse of nerve growth factor triggers long-term neuronal excitability through sodium channel gene induction. Neuron 14:607–611. 49. Toullec, D., P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Loriolle, et al. 1991. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266:15771–15781. 50. Wood, K. W., H. Qi, G. D’Arcangelo, R. C. Armstrong, T. M. Roberts, and S. Halegoua. 1993. The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction. Proc. Natl. Acad. Sci. USA 90:5016–5020. 51. Wooten, M. W., G. Zhou, M. L. Seibenhener, and E. S. Coleman. 1994. A role for zeta protein kinase C in nerve growth factor-induced differentiation of PC12 cells. Cell Growth Differ. 5:395–403.
