Proc. Ntatl. Acadil. Sci. U.SA Vol. 93, pp. 1113-1118, February 1996 Neurobiology Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels (epidermal growth factor receptor/Ras/mitogen-activated protein kinase) LAURA B. ROSEN*t AND MICHAEL E. GREENBERG*t§ *Division of Neuroscience, Department of Neurology, Children's Hospital, and tProgram in Biological and Biomedical Sciences and :Department of Neurobiology, Harvard Medical School, Boston, MA t)2115 Comminicated by Gerald D. Fischbach, Harvard Medical School, Boston, MA, October 2, 1995 ABSTRACT To understand the mechanisms by which elec- number of different signaling molecules that associate with trical activity may generate long-term responses in the nervous activated growth factor receptors (15, 16). SH2 domains bind system, we examined how activation of voltage-sensitive calcium to phosphorylated tyrosine residues and adjacent amino acid channels (VSCCs) can stimulate the Ras/mitogen-activated sequences, which determine the specificity of the interaction. protein kinase (MAPK) signaling pathway. Calcium influx One class of signaling molecule that inducibly binds to through L-type VSCCs leads to tyrosine phosphorylation of the growth factor receptors is the adaptor protein, which lacks adaptor protein Shc and its association with the adaptor protein catalytic moieties but mediates protein-protein interactions Grb2, which is bound to the guanine nucleotide exchange factor via modular domains such as SH2 domains. One of the adaptor Sosi. In response to calcium influx, Shc, Grb2, and Sosl induc- proteins that inducibly associates with tyrosine-phosphory- ibly associate with a 180-kDa tyrosine-phosphorylated protein, lated growth factor and cytokine receptors is the SH2/collagen which was determined to be the epidermal growth factor receptor protein (Shc) (17). Shc is itself also inducibly phosphorylated (EGFR). Calcium influx induces tyrosine phosphorylation of the on tyrosine in response to growth factor and cytokine stimu- EGFR to levels that can activate the MAPK signaling pathway. lation (18-22), which creates a consensus binding site (pYXN) Thus, ion channel activation stimulates growth factor receptor that is recognized by another SH2 domain-containing adaptor signal transduction. protein, growth factor receptor binding protein 2 (Grb2) (23). Grb2 contains, in addition to its SH2 domain, two SH3 Calcium influx into neurons is the critical transducer of domains that mediate its interaction with proline-rich se- electrical input into biochemical output (1). A wide range of quences in the Ras guanine nucleotide exchange factor (GEF) neurotransmitter receptors and second messenger systems termed mSosl (24-28). Induction of Grb2-Sosl association have been shown to regulate the influx of extracellular calcium with Shc through Y317 is a potential mechanism for Ras through their effects on voltage-sensitive calcium channels activation in response to growth factors and cytokine stimu- (VSCCs) and other ion channels in a process termed neuro- lation (29-35). modulation (2-4). However, the mechanisms by which calcium The parallels we found previously between calcium and influx elicits long-term neuronal responses are less clear. growth factor activation of MAPK suggested that tyrosine Studies of the biochemical responses generated in response phosphorylation might be involved in calcium activation of to calcium influx have focused on activation of cytoplasmic Ras. We report here that calcium influx upon activation of signaling molecules that directly bind calcium or calcium-cal- VSCCs leads to tyrosine phosphorylation of Shc and its modulin (CaM) complexes, such as calcium-CaM-dependent association with Grb2 and Sosl. In addition, this signaling adenylate cyclases and protein kinases (5). Calcium influx also complex inducibly associates with the epidermal growth factor leads by indirect mechanisms to activation of the ubiquitous receptor (EGFR), which is phosphorylated on tyrosine in mitogen-activated protein kinase (MAPK) pathway (6, 7), response to calcium influx to a level that is sufficient to lead which is a critical intermediate in long-term cellular responses to downstream MAPK activation. Our results demonstrate such as proliferation and differentiation (8-10). Recently, we that growth factor receptor signal transduction is activated in have demonstrated that stimulation of the MAPK pathway in response to VSCC stimulation, which may be an important response to calcium influx through L-type VSCCs involves biochemical mechanism by which neuronal activity can gen- activation of the small guanine nucleotide binding protein Ras erate long-term cellular responses. (11), a protooncogene product that mediates MAPK activa- tion in response to a wide variety of mitogens, cytokines, and MATERIALS AND METHODS trophic factors such as nerve growth factor (NGF) (8-10). To understand the mechanisms by which electrical activity may Materials. EGF was from Collaborative Biomedical Prod- initiate long-term responses in the ner.vous system, we exam- ucts (Bedford, MA), and NGF was purified from mouse ined how calcium influx leads to activation of Ras. salivary glands as described (36). Nifedipine was from Sigma. Growth factors initiate signaling processes that lead to Ras Anti-Trk antibodies were the generous gift of David Kaplan activation by binding to transmembrane receptors that contain (67). Other antibodies were obtained from the following intrinsic tyrosine kinase activity or, in the case of cytokine vendors: anti-phosphotyrosine [Tyr(p)] monoclonal antibody receptors, that are associated through their cytoplasmic do- (mAb) 4G10 from Upstate Biotechnology (Lake Placid, NY); mains with nontransmembrane protein tyrosine kinases such anti-Tyr(p) mAb PY20 from ICN; anti-Shc polyclonal antibody as the Src family members (12-14). Ligand binding induces (pAb) and mAb from Transduction Laboratories (Lexington, receptor dimerization and autophosphorylation on tyrosine KY); anti-Grb2 mAb from Upstate Biotechnology; anti-Grb2 residues. These phosphorylated tyrosines create binding sites for Src homology 2 (SH2) domains, which are present in a Abbreviations: VSCC, voltage-sensitive calcium channel; CaM, cal- modulin; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; GEF, guanine nucleotide exchange factor; EGF, epi- The publication costs of this article were defrayed in part by page charge dermal growth factor; EGFR, EGF receptor; mAb, monoclonal payment. This article must therefore be hereby marked 'advertisement" in antibody; pAb, polyclonal antibody. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1113 Downloaded by guest on October 8, 2021 1114 Neurobiology: Rosen and Greenberg Proc. Natl. Acad. Sci. USA 93 (1996) pAb from Santa Cruz Biotechnology (Santa Cruz, CA); anti- B IP: Grb2 Sosl pAb from Upstate Biotechnology; anti-EGFR pAb from A IP: Shc Upstate Biotechnology; anti-ErbB2 from Oncogene Science; Blot: C K N E Blot: C KN E goat anti-mouse pAb from Calbiochem; and rabbit anti-sheep i -84 P .Ty. 1S -84 pAb from Pierce. P-Tyr - -63 -63 Cell Culture and Stimulation. PC12 cells were obtained = : 0 w -53 from Simon Halegoua (68) and cultured on 100-mm tissue -35 : ,* i,-53-35 culture dishes (Falcon) in DMEM (GIBCO) supplemented with 10% heat-inactivated horse serum and 5% fetal bovine -32 -195 serum (GIBCO) in a humidified incubator with 10% C02/ Sosl -- - 90% air. Cell membranes were depolarized by addition of an Grb2 isosmotic solution of KCl (or NaCJ control) to a final concen- tration of 50 mM as described (11). _ __d -63 -32 Immunoprecipitation and Immunoblotting. Cells were lysed Shc __w -535 Grb2 _ _ in HNTG buffer [50 mM Hepes, pH 7.5/50 mM NaCl/1% Triton X-100/10% glycerol (vol/vol)/1.5 mM MgCl2/1 mM EDTA/10 mM sodium pyrophosphate/1 mM Na3VO4/100 FIG. 1. Calcium induction of Shc tyrosine phosphorylation and mM NaF/30 mM 2-(p-nitrophenyl) phosphate/I mM phenyl- Grb2 association. (A) PC12 cells were incubated with 50 mM NaCl methylsulfonyl fluoride/10 ,ug of aprotinin per ml/10 jig of control solution (lane C), 50 mM KCI (lane K), 100 ng of NGF per ml leupeptin per mlI and centrifuged at 10,000 x g for 15 min. (lane N), or 10 ng of EGF per ml (lane E) for 5 min. Lysates were immunoprecipitated with anti-Shc pAb. Washed immunoprecipitates Supernatants were mixed with primary antibody and rocked at (IP) were separated by PAGE and transferred to nitrocellulose for 4°C for 1-4 h. Secondary antibody and protein A-Sepharose immunoblotting with a mixture of anti-Tyr(p) antibodies 4G10 and (Calbiochem) were added for an additional 1-2 h. Immuno- PY20, anti-Grb2 mAb (Middle), or anti-Shc mAb (Bottom). Positions precipitates were washed three times in HNTG and resus- of migration of prestained molecular size markers (kDa) (Sigma) are pended in 2x Laemmli sample buffer (80 mM Tris-HCI, pH shown. Arrows indicate phosphorylated Shc isoforms of 48 and 56 6.8/15% glycerol/2% SDS/0.01% bromphenol blue/10% kDa. (B) PC12 cells were treated as in A. Lysates were immunopre- 2-mercaptoethanol). Precipitated proteins were separated by cipitated with a pAb against Grb2, and precipitated proteins were SDS/PAGE, transferred to nitrocellulose, and analyzed by analyzed by immunoblotting with anti-Tyr(p) antibodies (Top), anti- Western blotting as described (11). Antibody binding was Sosl pAb (Upstate Biotechnology; Middle), or anti-Grb2 mAb (Bot- tom) to confirm that equal levels