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Calcium Influx Activates Extracellular-regulated Kinase/Mitogen-activated Protein Kinase Pathway through a Calmodulin-sensitive Mechanism in PC12 Cells

1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês

10.1074/jbc.274.1.75

ISSN

1083-351X

Autores

Joaquim Egea, Carme Espinet, Joan X. Comella,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Evidence suggests that membrane depolarization is able to promote neuronal survival through a sustained, although moderate, increase in the intracellular calcium. We have used the PC12 cell line to study the possible intracellular pathways that can be activated by calcium influx. Previously, we observed that membrane depolarization-induced calcium influx was able to activate the extracellular-regulated kinase (ERK)/mitogen-activated protein kinase pathway and most of this activation was calmodulin-dependent. We demonstrated that a part of the ERK activation is due to the phosphorylation of the epidermal growth factor receptor. Here, we show that both the epidermal growth factor receptor phosphorylation and the Shc-Grb2-Ras activation are not calmodulin-modulated. Moreover, dominant negative mutant Ha-ras (Asn-17) prevents the activation on ERKs by membrane depolarization, suggesting that Ras and calmodulin are both necessaries to activate ERKs by membrane depolarization. We failed to observe any significant induction and/or modulation of the A-Raf, B-Raf or c-Raf-1 kinase activities, thus suggesting the existence of a MEK kinase different from the classical Raf kinases that directly or indirectly can be modulated by Ca2+/calmodulin. Evidence suggests that membrane depolarization is able to promote neuronal survival through a sustained, although moderate, increase in the intracellular calcium. We have used the PC12 cell line to study the possible intracellular pathways that can be activated by calcium influx. Previously, we observed that membrane depolarization-induced calcium influx was able to activate the extracellular-regulated kinase (ERK)/mitogen-activated protein kinase pathway and most of this activation was calmodulin-dependent. We demonstrated that a part of the ERK activation is due to the phosphorylation of the epidermal growth factor receptor. Here, we show that both the epidermal growth factor receptor phosphorylation and the Shc-Grb2-Ras activation are not calmodulin-modulated. Moreover, dominant negative mutant Ha-ras (Asn-17) prevents the activation on ERKs by membrane depolarization, suggesting that Ras and calmodulin are both necessaries to activate ERKs by membrane depolarization. We failed to observe any significant induction and/or modulation of the A-Raf, B-Raf or c-Raf-1 kinase activities, thus suggesting the existence of a MEK kinase different from the classical Raf kinases that directly or indirectly can be modulated by Ca2+/calmodulin. intracellular free Ca2+ concentration voltage-gated Ca2+channel mitogen-activated protein phosphatidylinositol 3-kinase high level of K+ nerve growth factor epidermal growth factor EGF receptor bisindolylmaleimide I phorbol 12-myristate 13-acetate extracellular-regulated kinase MAPK/ERK kinase calmodulin CaM-dependent protein kinase polyacrylamide gel electrophoresis Tris-buffered saline anti-phosphotyrosine glutathione S-transferase l-α-phosphatidylinositol tropomyosin receptor kinase protein kinase C phospholipase C 4-morpholinepropanesulfonic acid. Several studies have reported that chronic depolarization of plasma membrane prevent the cell death that occurs after deprivation of neurotrophic factors in many populations of neurons in culture. This effect is mediated by a sustained increase of the intracellular-free Ca2+ concentration ([Ca2+]i)1that enters the cell through voltage-gated calcium channels (VGCC) (1Collins F. Lile J.D. Brain Res. 1989; 502: 99-108Crossref PubMed Scopus (107) Google Scholar, 2Collins F. Schmidt M.F. Guthrie P.B. Kater S.B. J. Neurosci. 1991; 11: 2582-2587Crossref PubMed Google Scholar, 3Larmet Y. Dolphin A.C. Davies A.M. Neuron. 1992; 9: 563-574Abstract Full Text PDF PubMed Scopus (72) Google Scholar, 4Franklin J.L. Sanz-Rodriguez C. Juhasz A. Deckwerth T.L. Johnson E.M. J. Neurosci. 1995; 15: 643-664Crossref PubMed Google Scholar). E. M. Johnson's laboratory conceptualized this phenomenon in the "Ca2+ set-point hypothesis," which postulates that moderate increases in the [Ca2+]i (less than 100 nm above the basal values) can promote the survival of neurons in culture even in the absence of trophic support (for review, see Ref. 5Franklin J.L. Johnson E.M. Trends Biochem. Sci. 1992; 15: 500-508Google Scholar). However, the molecular basis relevant for the depolarization-induced neuronal survival have been shown to be controversial and remain to be elucidated (6D'Mello S.R. Borodezt K. Soltoff S.P. J. Neurosci. 1997; 17: 1548-1560Crossref PubMed Google Scholar, 7Miller T.M. Tansey M.G. Johnson Jr., E.M. Creedon D.J. J. Biol. Chem. 1997; 272: 9847-9853Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Soler R.M. Egea J. Mintenig G.M. Sanz-Rodriguez C. Iglesias M. Comella J.X. J. Neurosci. 1998; 18: 1230-1239Crossref PubMed Google Scholar). One possible mechanism by which membrane depolarization could promote survival is by activating signaling pathways similar to those activated by neurotrophic factors. Among these, the Ras/MAP kinase and the phosphatidylinositol 3-kinase (PI 3-kinase) pathways have been shown to be the most relevant (9Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (905) Google Scholar, 10Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (545) Google Scholar). Although membrane depolarization and subsequent increase in [Ca2+]i is not able to promote cell survival or differentiation in PC12, in low serum medium some effects of increased [Ca2+]i have been described. For example, membrane depolarization induced by adding KCl (high-K+) to the culture medium is able to preserve priming and preexisting neurites induced by NGF treatment (11Teng K.K. Greene L.A. J. Neurosci. 1993; 13: 3124-3135Crossref PubMed Google Scholar). It can also induce differentiation in cells sensitized with levels of NGF, which alone are insufficient to induce morphological differentiation (12Solem M. Mcmahon T. Messing R.O. J. Neurosci. 1995; 15: 5966-5975Crossref PubMed Google Scholar). Finally, it has been reported that potassium combined with Bay K induces long term survival and differentiation of PC12 cells (13Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar). Variations in the [Ca2+]i has been shown to be determinant in the regulation of the Ras/MAP kinase pathway through mechanisms not completely understood (14Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (599) Google Scholar, 15Finkbeiner S. Greenberg M.E. Neuron. 1996; 16: 233-236Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Rusanescuet al. (13Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar) demonstrated that an increase in [Ca2+]i directly or indirectly induces Shc tyrosine phosphorylation, which in turn associates with Grb2 and Sos, resulting in the activation of Ras. Moreover, membrane depolarization is able to induce tyrosine phosphorylation of the EGFR to a sufficient extent to activate the ERK/MAP kinase pathway (16Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar, 17Egea J. Espinet C. Comella J.X. J. Neurochem. 1998; 70: 2554-2564Crossref PubMed Scopus (33) Google Scholar, 18Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1998; 272: 24767-24770Abstract Full Text Full Text PDF Scopus (181) Google Scholar) and this activation seems to be necessary to activate this signaling pathway (18Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1998; 272: 24767-24770Abstract Full Text Full Text PDF Scopus (181) Google Scholar). Furthermore, PYK2, an intracellular tyrosine kinase related to the focal adhesion kinase, is also activated by increases in the [Ca2+]i that can in turn activate Ras through a Src-dependent pathway (19Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar). As Ca2+ ions enter the cytosol, they encounter a number of proteins that regulate their biochemical effects. Central among them is calmodulin (CaM), a small Ca2+-binding protein, which can bind up to four Ca2+ ions. After binding to Ca2+, CaM changes its conformation and it is able to regulate the activity of many different proteins. In several cell types, CaM has been shown to modulate Ras either directly or indirectly. Farnsworth et al. (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Felg L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (393) Google Scholar) have demonstrated that Ca2+ stimulation of Ras can be mediated by CaM through a Ras-GTP exchange factor which contains an IQ motif, referred to as Ras-GRF (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Felg L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (393) Google Scholar). Moreover, some CaM-binding Ras-like GTPases have been described (21Lee C.-H.J. Della N.G. Chew C.E. Zacks D.J. J. Neurosci. 1996; 16: 6784-6794Crossref PubMed Google Scholar, 22Wes P.D. Yu M.J. Montell C. EMBO J. 1996; 15: 5839-5848Crossref PubMed Scopus (72) Google Scholar), even though its functional activity on MAP kinase activation has not yet been tested. These include RIT, which is widely distributed in human tissues, and RIN, whose expression is unusually confined to the nervous system. However, none of these proteins has been shown to be present in the PC12 cell line. Additionally, CaM has been shown to be able to regulate several CaM-dependent protein kinases (CaM-K) being CaM K II and IV the best characterized. CaM-K IV has been linked to the activation of several MAP kinases (including JNK-1, p38, and to a lesser extent ERK2) (23Enslen H. Tokumitsu H. Stork P.J.S. Davis R.J. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10803-10808Crossref PubMed Scopus (261) Google Scholar). However, CaM-K IV has been shown to be absent in PC12 cells and therefore the involvement of this kinase in the activation of ERK MAP kinase is controversial (23Enslen H. Tokumitsu H. Stork P.J.S. Davis R.J. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10803-10808Crossref PubMed Scopus (261) Google Scholar). Recently, it has been suggested that PI 3-kinase can be involved in the regulation of the ERK MAP kinase pathway although its implication seems to be cell type- and ligand-specific (24Duckworth B.C. Cantley L.C. J. Biol. Chem. 1997; 272: 27665-27670Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). For example, in Swiss 3T3 fibroblasts, PI 3-kinase seems to regulate the prolonged activation of the ERK MAP kinases (25Grammer T.C. Blenis J. Oncogene. 1997; 14: 1635-1642Crossref PubMed Scopus (177) Google Scholar). Additionally, it has been demonstrated that integrin-dependent activation of the ERK MAP kinases is reverted by wortmannin and LY294002, two selective PI 3-kinase inhibitors (26King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar). Moreover, a report from the laboratory of Sacks (27Joyal J.L. Burks D.J. Pons S. Matter W.F. Vlahos C.J. White M.F. Sacks D.B. J. Biol. Chem. 1997; 272: 28183-28186Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) has shown that CaM is able to bind and modulate the activity of the PI 3-kinase. The relevance of these results in the PC12 cell system remains to be proved. Our laboratory has previously shown that membrane depolarization of PC12 cells (17Egea J. Espinet C. Comella J.X. J. Neurochem. 1998; 70: 2554-2564Crossref PubMed Scopus (33) Google Scholar) and chicken motoneurons (8Soler R.M. Egea J. Mintenig G.M. Sanz-Rodriguez C. Iglesias M. Comella J.X. J. Neurosci. 1998; 18: 1230-1239Crossref PubMed Google Scholar) is able to activate the ERK MAP kinase pathway through a Ca2+/CaM-dependent mechanism. In the present work, we have investigated more precisely the level at which CaM is acting on the Ras/MAP kinase pathway. We show here that this modulation is located lower to Ras but upstream of MEK. Moreover, our results suggest that CaM action is independent of the classical forms of Raf (c-Raf-1, A-Raf, or B-Raf) indicating the existence of a MEK kinase, different from Raf and activated by a Ras-dependent mechanism after membrane depolarization, that would be regulated directly or indirectly by Ca2+/CaM. PC12 cells were grown on 75-cm2 tissue culture dishes (Corning) in Dulbecco's modified Eagle's medium supplemented with 6% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 6% heat-inactivated horse serum (Life Technologies, Inc.). Medium was further supplemented with 10 mm Hepes. The M-M17–26 PC12 subline was grown in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 10% heat-inactivated horse serum (Life Technologies, Inc.). Culture medium from two cell lines was further supplemented with 20 units/ml penicillin and 20 μg/ml streptomycin. Cells were maintained at 37 °C in a saturating humidified atmosphere of 95% air and 5% CO2. For experiments, PC12 and M-M17–26 cells were allowed to proliferate in polyornithine precoated tissue culture dishes (Corning) until they reached 80% confluence. Before acute stimulation with NGF (100 ng/ml), KCl (75 mm), or PMA (1.6 μm), cells were washed three times and cultured for an additional 15–20 h in serum-free medium. Before acute stimulations, the indicated cultures were exposed to different protein inhibitors: the CaM inhibitors calmidazolium chloride (Calbiochem-Novabiochem Corp., San Diego, CA), trifluoperazine dimaleate (Calbiochem-Novabiochem Corp.), W5 and W7 (Sigma), and W12 and W13 (Sigma); the PKC inhibitor BIM I (Calbiochem-Novabiochem Corp.); the MEK inhibitor PD98059 (Calbiochem-Novabiochem Corp.); or the PI 3-kinase inhibitor LY 294002 (Calbiochem-Novabiochem Corp.). After stimulation, cells were rinsed rapidly in ice-cold phosphate-buffered saline at pH 7.2 and solubilized at 4 °C in 0.4 ml of lysis buffer (see below). After 15 min of incubation on ice, cells were scraped from the dishes and cell lysates were orbitally rotated for 30 min at 4 °C. Nuclei and cellular debris were removed by microcentrifuge centrifugation at 10,000 ×g and 4 °C for 15 min. Protein concentration in the supernatant was quantified by a modified Lowry assay as described by the provider (Bio-Rad DC protein assay). Western blot assay was performed with immunoprecipitates or cell lysates by resolving the proteins in SDS-polyacrylamide gels. The proteins were transferred onto polyvinylidene difluoride Immobilon-P transfer membrane filters (Millipore, Bedford, MA) using a Pharmacia semidry Trans-Blot (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. Antibodies against the phosphorylated forms of ERK1 and ERK2, MEK1/2 and Akt (New England Biolabs, Inc., Beverly, MA), pan-ERK and c-Raf-1 (Transduction Laboratories, Lexington, KY), pan-MEK (New England Biolabs, Inc.), MEK1 (UBI, Lake Placid, NY), PLCγ (Transduction Laboratories), EGFR, A-Raf and B-Raf (Santa Cruz Biotechnology Inc., Santa Cruz, CA), or pan-Shc (Transduction Laboratories) were used according to the supplier instructions. After incubation with specific peroxidase-conjugated secondary antibodies, membranes were developed with an enhanced chemiluminescence Western blotting detection system (Pierce) Immunoprecipitation of Shc, PLCγ, or c-Raf-1 (Transduction Laboratories) and EGFR, A-Raf, or B-Raf (Santa Cruz Biotechnology Inc.) was performed with specific antibodies according to the supplier's instructions. Immunoprecipitated proteins were electrophoresed, transferred, and detected essentially as described above. To detect Grb2 association in Shc immunoprecipitates, membranes were blocked with TBS-T20 (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.05% Tween 20) containing 5% nonfat milk, probed with a polyclonal anti-Grb2 antibody diluted in TBS-T20 containing 0.2% nonfat milk and, finally, incubated with a specific peroxidase-conjugated secondary antibody. To detect tyrosine-phosphorylated PLCγ and EGFR, membranes were blocked with TBS-T20 containing 5% bovine serum albumin, probed with the 4G10 anti-phosphotyrosine monoclonal antibody (anti-Tyr(P)) and incubated with a specific peroxidase-conjugated secondary antibody. To immunoprecipitate the p85 subunit of the PI 3-kinase, extracts from NGF-treated or depolarized PC12 cells were subjected to immunoprecipitation overnight at 4 °C with the anti-Tyr(P) antibody 4G10 (1/100). Immunocomplexes were precipitated with protein A-Sepharose coupled to rabbit anti-mouse polyclonal antibody. p85 was detected using an specific anti-p85 antibody (UBI) as described by the supplier. MEK in vitrokinase assay was performed in MEK immunoprecipitates by using recombinant GST-ERK2 (UBI) and [γ-32P]ATP (Amersham Pharmacia Biotech) as substrates. After treatment, cells were lysed with lysis buffer (1% Nonidet P-40, 0.25% deoxycholate, 50 mm Tris, pH 7.5, 1 mm EGTA, 50 mmβ-glycerophosphate, 150 mm NaCl, 25 mm NaF, 1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 2 mmbenzamidine, and 20 μg/ml leupeptin). After the removal of nuclei and cellular debris, cell lysates were precleared for 1 h at 4 °C with 20 μl (v/v) of protein A-Sepharose. Five hundred μg of protein of the supernatant was transferred to a new tube, and the anti-MEK1 antibody was added (1/250) (UBI). After 2 h at 4 °C, immunocomplexes were precipitated with 40 μl (v/v) of protein A-Sepharose for an additional 1 h at 4 °C. Precipitates were washed three times with lysis buffer and three more times with assay kinase buffer (20 mm MOPS, pH 7.2, 1 mmdithiothreitol, 5 mm EGTA, 25 mmβ-glycerophosphate, 1 mm sodium orthovanadate). Precipitates were resuspended in 50 μl (final volume) of assay kinase buffer supplemented with 100 μm ATP, 15 mmMgCl2, 6 μCi of [γ-32P] ATP (3000 Ci/mmol) (Amersham Pharmacia Biotech), and 400 ng of GST-ERK2 (UBI). Kinase assay was allowed to proceed for 30 min at 30 °C. Reaction was stopped with 5× SDS-PAGE sample buffer, and products were separated by SDS-PAGE. After drying the gel, the phosphorylation signal was quantified on a PhosphorImager (Boehringer Mannheim). Radioactive spots were also detected by autoradiography by exposing the TLC plate to Fuji medical x-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan) overnight at –70 °C. Raf kinase activity was measured by using wild-type MEK1 (Santa Cruz Biotechnology Inc.) and [γ-32P] ATP (Amersham Pharmacia Biotech) as substrates. After treatments, cells were lysed with lysis buffer (1% Triton X-100, 20 mm Tris, pH 7.5, 2 mm EDTA, 50 mm β-glycerophosphate, 137 mm NaCl, 25 mm NaF, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 2 mm benzamidine, and 20 μg/ml leupeptin). After the removal of nuclei and cellular debris, cell lysates were precleared for 1 h at 4 °C with 20 μl (v/v) of protein G-Sepharose. Seven hundred and fifty μg of protein of the supernatant was transferred to a new tube, and 1 μg of anti-c-Raf-1 antibody (Transduction Laboratories), anti-B-Raf antibody, or anti-A-Raf antibody (Santa Cruz Biotechnology Inc.) was added. After 2 h at 4 °C, immunocomplexes were precipitated with 40 μl (v/v) of protein G-Sepharose for an additional 1 h at 4 °C. Precipitates were washed three times with lysis buffer and three more times with assay kinase buffer (25 mm Hepes, pH 7.5, 1 mm dithiothreitol, 1 mm EGTA, 25 mmβ-glycerophosphate, 1 mm sodium orthovanadate). Precipitates were resuspended in 25 μl (final volume) of assay kinase buffer supplemented with 100 μm ATP, 50 mmMgCl2, 6 μCi of [γ-32P] ATP (3000 Ci/mmol) (Amersham Pharmacia Biotech), and 120 ng of wild type MEK1 (Santa Cruz Biotechnology Inc.). Kinase assay was allowed to proceed for 20 min at 30 °C. Reaction was stopped with 5× SDS-PAGE sample buffer, and products were separated by SDS-PAGE. After drying the gel, the phosphorylation signal was quantified and detected as described above. After stimulation, cells were solubilized in 1% Nonidet P-40 buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 2 mmbenzamidine, and 20 μg/ml leupeptin). Nuclei and debris were removed by centrifugation, and approximately 750 μg of protein were subjected to immunoprecipitation overnight at 4 °C with the anti-Tyr(P) antibody, 4G10. Immunocomplexes were collected with protein A-Sepharose preconjugated with a rabbit anti-mouse IgG antibody and sequentially washed with lysis buffer, LiCl buffer (100 mm Tris, pH 7.5, 0.5 m LiCl, 1 mm EDTA, and 1 mmsodium orthovanadate), and TNE buffer (25 mm Tris, pH 7.5, 100 mm NaCl, and 1 mm EDTA). Immunocomplexes were incubated with a mixture of l-α-phosphatidylinositol and l-α-phosphatidyl-l-serine (final concentration 0.5 mg/ml each) and 10 μCi of [γ-32P] ATP. Incubation was allowed to proceed for 20 min at room temperature. Phosphorylated lipids were then extracted and resolved by TLC usingn-propanol:H2O:acetic acid (66:33:2, v:v:v) as solvent. Radioactive spots were detected by autoradiography by exposing the TLC plate to Fuji medical x-ray film (Fuji Photo Film Co. Ltd.) overnight at –70 °C. Ras activity was measured with a non-radioactive method as described previously (28de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). Briefly, treated cells were solubilized for 15 min in lysis buffer containing 25 mm Tris, pH 7.5, 5 mm EGTA, 15 mmNaCl, 5 mm MgCl2, 1% Triton X-100, 1%N-octyl-β-d-glucopyranoside, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 2 mmbenzamidine, and 20 μg/ml leupeptin. Nuclei and cellular debris were removed, and 50 μg of the recombinant GST-RBD protein previously coupled to glutathione-Sepharose (Amersham Pharmacia Biotech) were added to approximately 750 μg of protein. Protein complexes were allowed to form for 2 h at 4 °C. Precipitates were washed three times with lysis buffer withoutN-octyl-β-d-glucopyranoside and once with phosphate-buffered saline. Finally precipitates were resuspended with SDS-PAGE loading buffer and denatured proteins were loaded in a 12% SDS-PAGE. Immunodetection was done using an anti-pan-Ras antibody (Oncogene Research Products, Cambridge, MA) and a anti-mouse IgG coupled to horseradish peroxidase as a secondary antibody. Blots were developed with the enhanced chemiluminescence Western blotting detection system described above. The rest of biochemicals were obtained from Sigma. Anti-Grb2 was a gift from Dr. J. Ureña (University of Barcelona, Barcelona, Spain), anti-pan-Ras was from Dr. O. Bachs and Dr. N. Agell (University of Barcelona, Barcelona, Spain), anti-EGFR was from Dr. G. Capellà and Dr. C. Garcı́a (Hospital de Sant Pau, Barcelona, Spain), and anti-Tyr(P) (4G10) were from Dr. D. Martin-Zanca (CSIC-University of Salamanca, Salamanca, Spain). The GST-RBD construct was obtained from Dr. F. McKenzie (State University of New York, Stony Brook, NY) through Dr. O. Bachs and Dr. N. Agell. The PC12 subline M-M17–26, kindly provided by Dr. G. M. Cooper (Harvard Medical School, Boston, MA) through Dr. A. Aranda (CSIC, Madrid, Spain), was obtained after transfection with the dominant negative mutant Ha-ras (Asn-17). 7 S NGF was prepared in our laboratory from salivary glands as described previously (29Mobley W.C. Schenker A. Shooter E.M. Biochemistry. 1976; 15: 5543-5552Crossref PubMed Scopus (524) Google Scholar). We have previously reported that ERK MAP kinase activation induced by high-K+ in PC12 cells is specifically blocked by the CaM antagonist W13, but not by its structural analogue W12, which is about 5 times less potent (17Egea J. Espinet C. Comella J.X. J. Neurochem. 1998; 70: 2554-2564Crossref PubMed Scopus (33) Google Scholar, 30Hidaka H. Sasaki Y. Tanaka T. Endo T. Ohno S. Fujii H. Nagata Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4354-4357Crossref PubMed Scopus (559) Google Scholar,31Hidaka H. Tanaka T. Methods Enzymol. 1983; : 185-193Crossref PubMed Scopus (124) Google Scholar) (Fig. 1 A). In the present report, this result has been extended to other CaM inhibitors that include W7/W5 (30Hidaka H. Sasaki Y. Tanaka T. Endo T. Ohno S. Fujii H. Nagata Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4354-4357Crossref PubMed Scopus (559) Google Scholar, 32Tanaka T. Hidaka H. J. Biol. Chem. 1980; 255: 11078-11080Abstract Full Text PDF PubMed Google Scholar) (Fig. 1 B), calmidazolium (33Gietzen K. Xu Y.H. Galla H.J. Bader H. Biochem. J. 1982; 207: 637-640Crossref PubMed Scopus (12) Google Scholar, 34Hu J. el-Fakahany E.E. Neuroreport. 1993; 4: 198-200Crossref PubMed Scopus (14) Google Scholar) (Fig. 1 C), and trifluoperazine (35Massom L. Lee H. Jarrett H.W. Biochemistry. 1990; 29: 671-681Crossref PubMed Scopus (73) Google Scholar, 36Tatsuta M. Iishi H. Baba M. Yano H. Uehara H. Nakaizumi A. Iseki K. Cancer Lett. 1996; 107: 179-185Crossref PubMed Scopus (6) Google Scholar) (Fig.1 D). In all cases, these inhibitors showed a similar effect to that elicited by W13, i.e. when PC12 cell were pretreated with the inhibitors, they prevented the activation of the ERK MAP kinase induced by high-K+ (Fig. 1). The blockade of high-K+-induced ERK activation was dose-dependent (data not shown) and specific, since structural homologues (W5 and W12) of active inhibitors (W7 and W13, respectively) were not able to block the ERK activation (Fig. 1,A and B). Moreover, prevention of ERK activation exerted by the CaM inhibitors was not mediated by an alteration of the calcium currents after membrane depolarization, i.e. these drugs did not alter the kinetics of Ca2+ entry into the cytoplasm (data not shown) (8Soler R.M. Egea J. Mintenig G.M. Sanz-Rodriguez C. Iglesias M. Comella J.X. J. Neurosci. 1998; 18: 1230-1239Crossref PubMed Google Scholar, 17Egea J. Espinet C. Comella J.X. J. Neurochem. 1998; 70: 2554-2564Crossref PubMed Scopus (33) Google Scholar). It has been previously reported that transactivation of receptors with tyrosine kinase activity such the EGFR in PC12 cells by membrane depolarization seems to be necessary to reach a complete activation of the ERK MAP kinase pathway (16Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar, 17Egea J. Espinet C. Comella J.X. J. Neurochem. 1998; 70: 2554-2564Crossref PubMed Scopus (33) Google Scholar, 18Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1998; 272: 24767-24770Abstract Full Text Full Text PDF Scopus (181) Google Scholar). These data suggested us the possibility that CaM inhibitors may exert its effects on the high-K+-induced activation of ERKs indirectly through the blockade of the kinase activity of the EGFR. To test this hypothesis, the EGFR was immunoprecipitated from 2- and 5-min depolarized cells pretreated or not with the CaM antagonists W12 and W13. To study the kinase activity of the receptor, immunoprecipitates were analyzed on Western blot with an anti-Tyr(P) antibody. As shown in Fig.2, high-K+ was able to activate the tyrosine kinase activity of the EGFR although to a much lesser extent than EGF. However, in cell lysates where the ERK phosphorylation due to membrane depolarization was completely prevented by W13 (data not shown), neither W13 nor W12 pretreatment was able to modify the level of tyrosine phosphorylation of the receptor (Fig. 2). Therefore, the observed lack of ERK phosphorylation in the W13-treated cultures after membrane depolarization could not be attributed to an inhibition of the kinase activity of the EGFR. The ERK MAP kinase cascade is usually initiated by the interaction of trophic factors with their corresponding tyrosine kinase receptors resulting in the autophosphorylation of the receptor. Phosphorylated tyrosine kinase receptors activate Ras by a mechanism that requires the tyrosine phosphorylation of Shc (for review, see Ref. 9Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (905) Google Scholar). The ability of Shc to activate Ras is mediated by the association of Shc to Grb2 and Sos. It has been previously reported that depolarization-induced ERK activation can result from a direct depolarization-induced phosphorylation of tyrosine kinase receptors such as the EGFR (18Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1998; 272: 24767-24770Abstract Full Text Full Text PDF Scopus (181) Google Scholar). Nevertheless, the activation of EGFR was not sensitive to W13 (see above). Shc has also been reported to play a central role in the Ca2+-induced ERK activation after membrane depolarization (15Finkbeiner S. Greenberg M.E. Neuron. 1996; 16: 233-236Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). We have analyzed the possibility that the W13 CaM antagonist can modulate the ERK activity through the function of Shc. To test this possibility, we have studied the effects of CaM antagonists over the tyrosine phosphorylation of Shc and its association to Grb2 after membrane depolarization. For this, PC12 cells were pretreated with W13 and then the cells were depolarized for 2 and 5 min with 75 mm KCl. The structurally related W12 homologue was included in the experiments to assess the specificity of the W13 effects. Extracts were subjected to immunoprecipitation with an anti-Shc antibody that recognizes the 66-, 52-, and 46-kDa isoforms of this protein. Immunoprecipitates were resolved in SDS-PAGE, b

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