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ICANCERRESEARCH57.3168-3173, August 1. 19971 Src Tyrosine Kinase Mediates Stimulation of Raf-1 and Mitogen-activated Protein Kinase by the Tumor Promoter Thapsigargin' Tsung-Shu Oliver Chao,2 Mark Abe, Marc B. Hershenson, Ignatius Gomes, and Marsha Rich Rosner@ The Ben May Institute for Cancer Research and Departments of Pharmacological and Physiological SciencesiT-S. 0. C., 1.G., M. R. R.j and Pediatrics fM. A., M. B. H.). University of Chicago. Chicago. Illinois 60637 ABSTRACT signal-regulated kinase, or ERK). This activation is independent of @ protein kinase C or Ca2 influx (2) but requires the presence of Raf-l Thapsigargin is a non-phorbol ester-type tumor promoter that elevates (7). The activation of MAP kinase can lead to the specific phospho the intracellular Ca2@ (Ca@2') levels by blocking the microsomal Ca2@ rylation of EGF receptor on threonine 669 (8, 9) as well as a subse ATPase. At present, the consequence of this Ca@2@increaseand the nature quent stimulation of a membrane-associated phosphatase (10). These of the tumorigenicity of thapsigargin still remain to be elucidated. Previ ously, we demonstrated that thapsigargin activates the mitogen-activated events appear to underlie the negative regulation of EGF receptor by protein (MAP) kinase via Ca@2' but independently of protein kinase C or both phorbol ester (11) and thapsigargin action (10, 12, 13). More Ca2―Influx.Here, we show that thapsIgar@n also rapidly stimulates the over, they suggest that the MAP kinase signaling pathway can play a Src tyrosine kinase. Transfection of a v-Src gene into a hippocampal cell role in mediating the cellular influence of thapsigargin. line (H19—7)rendersa constitutive activation of MAP kinase, whereas Activation of MAP kinase is a rapid response shared by diverse transfection of a kinase-deficient Src mutant blocks the activation by groups of growth modulators and tumor promoters. Numerous reports thapsigargin, suggesting that Src is required for the thapsigargin-induced have documented a well-conserved signaling pathway for MAP ki MAP kinase activation. Cotransfection of a doednant-inifibitory Raf-1 nase activation that requires a sequential activation of Grb2, Sos, Ras, and the v-Src genes into H19—7 cells results in an Inhibition of the Raf, and MEKS (reviewed in Ref. 14). It is also known that alternative otherwise constitutively elevated MAP kinase activity, suggesting that pathways for activation of MAP kinase exist in parallel (15, 16). Raf-1 is required for the Src-dependent activation of MAP kinase. Sinai lady, in the LA-90 cells, expression of a temperature-sensitive allele of Although MAP kinase appears to integrate a network of intracellular v-Src constitutively activates Raf.1 and MAP kinase, whereas expression signals, it is of importance to identify and distinguish how different of a dominant-Inhibitory Raf-1 mutant abolishes the MAP kinase activa extracellular stimuli transfer their signals to MAP kinase. tion Induced by either v-Src or thapsigargin treatment Together, these In this report, we focus our investigation on the action of thapsi results suggest that thapsigargin stimulates MAP kinase signaling via Src gargin that is related to MAP kinase signaling. We show here that and Raf-1. The activation of this Src-MAP kinase pathway suggests a thapsigargin rapidly stimulates the Src tyrosine kinase; the Src kinase biochemical mechanism for the tumorigenic nature of thapsigargin. activity is required for the thapsigargin-induced activation of Raf-1 and MAP kinase. Because various cellular components, such as cy INTRODUCTION toskeletal proteins, focal adhesion kinase, and GTPase activating protein, are substrates of Src tyrosine kinase (17), it is conceivable Thapsigargin, originally isolated from the plant Thapsia garganica that the stimulation of Src, as well as the subsequent activation of L. (Apiaceae), is a non-phorbol ester-type tumor promoter (1). It Raf-l and MAP kinase, may contribute to the molecular basis of the inhibits the microsomal Ca2@ ATPase, causing the Ca2@to leak out of tumorigenicity of thapsigargin. the internal stores and elevating the Ca@2@4levels(1, 2). The depletion @ of internal Ca2 stores is then followed by a subsequent “capacitative entry―of extracellular Ca2@ through surface channels (3, 4). The MATERIALS AND METHODS effect of thapsigargin on Ca2@ mobilization has been studied exten EGF (receptor grade) was purchased from Biomedical Technologies sively (3, 5); however, the molecular mechanism for its tumorigenic (Stoughton, MA). Thapsigargin and MBP were from Sigma Chemical Co. ity remains to be elucidated. Several lines of evidence have suggested Anti-p2lras monoclonal antibody (Yl3—259) and anti-c-Raf-l antibody were that phosphorylation events are essential in relaying thapsigargin from Santa Cruz Biotechnology, Inc. Anti-Src antibody (Ab327) was a gift action. For example, the transcriptional activation of the glucose from Dr. J. Brugge (ARID Pharmaceuticals, Cambridge, MA). Anti-MAP regulated protein GRP78 promoter by thapsigargin required both kinase antiserum (Ab283) was developed as described previously (2). The tyrosine and serine/threonine kinases (6). On the other hand, alkaline plasmid encoding the kinase-inactive MEK was a gift from Dr. G. Johnson phosphatase abolished the activity of thapsigargin-activated calcium (National Jewish Center for Immunology and Respiratory Medicine, Denver, influx factors from Jurkat cells to induce rapid chloride current in the CO), andplasmidsencodingv-Srcandkinase-inactiveSrcwerea gift fromD. oocytes (4). Foster (Hunter College, New York, NY). Previously, our laboratory demonstrated that thapsigargin-induced Cell Cultures. The H19—7cellswere generated from rat hippocampal neurons (18). The cells were conditionally immortalized by stable transfection release of Ca@2@activated MAP kinase (also known as extracellular with a temperature-sensitive SV4O large T antigen. H19—7cells were grown at 33°Cin DMEMJ1O% fetal bovine serum and kept under G4l8 selection Received 1/13/97: accepted 5/28/97. throughout the experiments (18, 19). Under these conditions, the cells are The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with undifferentiated and proliferate in response to serum. 18 U.S.C. Section 1734 solely to indicate this fact. The dominant-negative Raf-1 cell line Rl was generated by stably trans I This work was supported by NIH Grant CA 35541 (to M. R. R.) and a gift from the fecting the LA-90 cells with the mutant Raf vector p301—1(20). The LA-MNC Cornelius Crane Foundation. control cell line was generated by transfecting with the parental vector to 2 Current address: Synthélabo Research, Bagneux 92225, France. p301—1(pMNC).The LA-90 cells express a temperature-sensitive derivative 3 To whom requests for reprints should be addressed, at the Ben May Institute for Cancer Research, MC 6027, University of Chicago, 5841 South Maryland Avenue, of Src (20). Experiments with activated Src were performed by shifting the Chicago, IL 60637. Phone: (773) 702-1857; Fax: (773) 702-4634 or 6260; E-mail: cells to the permissive temperature (33°C)for10 mm; the control experiments [email protected]. were performed at the nonpermissive temperature (39°C;Refs.7 and 20). @@ 4 The abbreviations used are: Ca,2@, intracellular Ca2 MAP, mitogen-activated Transient Transfection of H19—7Hippocampal Cells. The H19—7cells protein; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; MEK, MAP kinase kinase; MBP, myelin basic protein; LPA, lysophosphatidic acid; BK, were transfected with plasmids encoding the hemagglutinin-tagged ERK2, bradykinin; PMSF, phenylmethylsulfonyl fluoride. kinase-deficient Src Arg295 (21), v-Src, and dominant-inhibitory Rafp3Ol and 3168 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1997 American Association for Cancer Research. THAPSIGARGIN ACTIVATES Src, Raf-l, AND MAP KINASE A C4(7,20)usingthecalciumphosphateprecipitationmethod.TheSrcand @ 4 ______________________________________________ Raf-l constructs were about 4-fold in excess of the epitope-tagged ERK2 construct. A plasmid encoding @3-galactosidase driven by a cytomegalovirus —0- Thaps promoter was cotransfected to normalize for transfection efficiency. After transfection, the cells were allowed to grow in fresh medium with 10% FBS for U 16—24 h. The plates containing the cells from the same transfection were @@ >1 pooled and replated to ensure homogeneous transfection efficiency (approxi :@ mately 15%). The replated cells were grown in fresh medium with 10% FBS @ :@ for 20—24hand subsequently starved for another 20—24h.The transfected 4 : cells were harvested for experiments within 60—72 h. @@@ 2 Sit KinaseAssay.Srctyrosmnekinaseactivitywasmeasuredbyphospho @ — rylation of rabbit muscle enolase (22, 23). Stimulated and control cells were @ :@ washedtwicewithPBSandthenlysedin50nmiTris,pH8.0;0.5%NP-40; @ 0 120 mt@i NaCl; 1 mM EDTA; 20 @tg/ml aprotinin; 200 mM Na3VO4; and 0.2 nmi c@5 PMSF. After removal of debris, the supematant was incubated with anti-Sec antibody (Ab327) for 30 mm. Protein A-Sepharose was added for another 30 mm. The mixtures were washed in radioimmunoprecipitation assay buffer and once in Tris-buffered saline (20 mrsi Tris, pH 7.5; 150 mM NaCl). The pellet was resuspended in 10 @.tlofkinase buffer (20 mai HEPES, pH 7.2; 5 mM @@@ 0 . 20 30 MnCl2;

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