Stimulation of Tyrosine Kinase Activity in Anti-phosphotyrosine ...

Vol. 266, No. 35, Issue of December 15, pp. 24126-24133,1991 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Stimulation of Tyrosine Kinase Activityin Anti-phosphotyrosine Immune Complexes of Swiss 3T3 Cell Lysates Occurs Rapidly after Addition of Bornbesin, Vasopressin, and Endothelin to IntactCells* (Received for publication, June 24, 1991)

Ian Zachary, James Sinnett-Smith, and Enrique Rozengurtl From the Imperial Cancer Research Fund, P. 0. Box 123, Lincoln’s Inn FieMs, London WC2A 3PX, United Kingdom

Treatment of quiescent Swiss 3T3 cells with the kinase C (PKC)’ (13-16). Bombesin has also recently been mitogenic peptides bombesin, vasopressin, endothelinl shown to stimulate the release and metabolism of arachidonic vasoactive intestinal contractor (VIC), and bradykinin acid in Swiss 3T3 cells (17). strikingly increased the initial rate of tyrosine phosBiochemical studies using permeabilized cells (16, 18, 19) phorylation measured in anti-phosphotyrosine immu- and membrane and solubilized receptor preparations (20-24) noprecipitates of a major band of M, 115,000 (p115) have demonstrated that bombesin and vasopressin act and two minor components of M, 90,000 and 75,000. through signal transduction pathways inwhich effector stimNeuropeptides increased the labeling of p l l 5 within ulation istightly coupled to activation of aGTP-binding seconds and with great potency; half-maximum con- protein. In addition, the receptors for bombesin in Swiss 3T3 centrations were 0.1, 0.2 and 0.3 nM for bombesin, cells (25, 26), and the human endothelins (27, 28) have revasopressin, and VIC, respectively. Immunoblotting cently been cloned and shown to belong to thefamily of GTPand peptide mapping showed that thepl15 band phosphorylated in anti-phosphotyrosine immunoprecipi- binding protein-linked receptors with seven hydrophobic dotates is identical to a major M , 115,000 substrate for mains (29). In contrast, receptors for polypeptide growth factors such neuropeptide-stimulated tyrosine phosphorylation in as platelet-derived growth factor (PDGF), possess intrinsic intact Swiss 3T3 cells. Furthermore, bombesin, vasoprotein tyrosine kinase activity (30). Tyrosine kinases and pressin, andVIC markedly increasedthe rateof phosphorylation of Raytide, a broad specificity tyrosine phosphorylation of cellular substrates are essential for the kinase peptide substrate, by decreasing (8 j : 1.3-fold) biological activity of this class of growth factor, as well as the apparentK,,, of the kinase for the substrate. Phor-many nonreceptor oncogenes (30-32), but its occurrence in bo1 12,13-dibutyrate and the Ca2+ionophore A23187 the action of neuropeptides is only beginning to be recognized had a weaker effect on tyrosine protein kinase activityin intact cells (33-36). Recently, we reported that bombesin, in immune complexes compared with bombesin. Fur- vasopressin, andendothelin rapidly increase tyrosine and thermore, down-regulationof protein kinaseC blocked serine phosphorylation of multiple substrates in quiescent the small effect of phorbol esters but did not impair Swiss 3T3 cells (36). The major substrates for neuropeptide bombesin-stimulated tyrosine kinase activity. These tyrosine phosphorylation appear to be unrelated to known results provide direct evidence for neuropeptide acti- targets for the PDGF receptor (36), including GTPase-activation of a tyrosine kinase in cell-free preparations vating protein, phosphatidylinositol 3’-kinase, and phosphoand identifya novel event in the action of this classof lipase C r (reviewed in Ref. 32). However, neither our studies growth factors in Swiss 3T3 cells. (36) nor those of other laboratories (33-35)showed that increased tyrosine phosphorylation by multiple neuropeptides was due to activation of a tyrosine protein kinase that could be measured in cell-free preparations. Neuropeptides stimulate DNA synthesis and proliferation The results presented here demonstrate that bombesin, in cultured cells and are implicated as growth factors in a vasopressin, the endothelin-related peptide, vasoactive intesvariety of biological processes, including embryogenesis, tissue tinal contractor (VIC), and bradykinin rapidly increase tyroregeneration, and tumorigenesis (1). In particular, several sine kinase activity measured in anti-phosphotyrosine imneuropeptides, including bombesin, vasopressin, the endothe- munoprecipitates of extracts from Swiss 3T3 cells. Further, lins, and bradykinin are potent mitogens for quiescent Swiss tyrosine phosphorylation in immune complexes and in intact 3T3 cells (2-6), a useful model system for the elucidation of cells occurs independently of PKC activation and mobilizasignal transduction pathways leading to cell proliferation (7). tion of Ca2+.These findings provide direct evidence for the These neuropeptides bind to specific, high affinity receptors existence of a novel event inthe action of multiple neuropep(8-12) and stimulate avariety of early biochemical responses tides, namely the stimulation of a tyrosine protein kinase (7, 12), including rapid hydrolysis of polyphosphoinositides, activity in Swiss 3T3 cells. mobilization of intracellular Ca2+ and activation of protein

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 To whom all correspondence should be addressed. Tel. 071-2693455; Fax: 071-430-1263.

The abbreviations used are: PKC, protein kinase C; anti-Tyr(P), anti-phosphotyrosine; DMEM, Dulbecco’s modified Eagle’s medium; p115, M. 115,000 phosphotyrosyl protein; PBu, phorbol 12,13-dibutyrate; PDGF, platelet-derived growth factor; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; VIC, vasoactive intestinalcontractor; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

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mM EDTA. The bands were then placed in the wells of a discontinEXPERIMENTALPROCEDURES uous 15% polyacrylamide gel and overlaid with 20 p1 of the same Cell Culture-Stock cultures of Swiss 3T3 fibroblasts were main- buffer containing 15% glycerol and 5 pg of Staphylococcus aureus V8 tained in Dulbecco'smodifiedEagle's medium (DMEM) supple- protease. mented with 10% fetal calf serum in a humidified atmosphere conWestern Blotting-Treatment of quiescent cultures of cells with taining 10% COz and 90% air a t 37 "C (2). Forexperimental purposes, factors, cell lysis, and immunoprecipitations were performed as decells were plated either in 33-mm Nunc Petri dishes a t lo5 cellsldish, scribed elsewhere. After SDS-PAGE, phosphotyrosyl proteins were or in 90-mm dishes at 5 X lo5 cells/dish in DMEM containing 10% transferred to Immobilon membranes (39). Membranes were blocked fetal calf serum and used after 6-8 days when the cells were confluent using 5% nonfat dried milk in phosphate-buffered saline, pH 7.2, and and quiescent (2). Immunoprecipitations-Quiescent cultures of cells were washed incubated for 3-5 h with the Py20 monoclonal anti-Tyr(P) antibody in phosphate-buffered saline containing 0.05% Tween-20 and 1 pg/ twice with DMEM, treated with peptide factors in 1 ml of DMEM as indicated, and lysed at 4 "C in 1 ml of a solution containing 10 mM ml antibody. Immunoreactive bands were visualized using lZ5I-labeled Tris/HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyro- sheep anti-mouse IgG. Down-regulation of PKC-PKC activity is extensively down-reguphosphate, 50 mM NaF, 100 p~ Na3V04,and 1% Triton X-100 (lysis lated in Swiss 3T3 fibroblasts by prolonged pretreatment with phorbol buffer). Lysates were clarified by centrifugation at 15,000 X g for 10 min and precleared by incubation with albumin-agarose for 1 h a t 12,13-dibutyrate (PBu) (12,41-44). In the present studies, confluent 4 "C. After removal of albumin-agarose by brief (10 s) centrifugation, and quiescent cultures were pretreated with 800 nM PBu for 40 h in the supernatantswere transferred to a fresh tube, and phosphotyrosyl conditioned medium. In some experiments, pretreated cells were proteins were immunoprecipitated for 3 h a t 4 "C with a monoclonal labeled with 32Pias described above also in the presence of 800 nM anti-phosphotyrosine (anti-Tyr(P))antibody coupled to agarose. Im- PBu. Conditioned medium is taken from cultures of Swiss 3T3 cells munoprecipitates were washed three times with lysis buffer and grown in DMEM with 10% fetal calf serum that have attained further analyzed as described under "Results" and in the figure quiescence and is therefore depleted of growth-promoting activity. Materials-Bombesin, vasopressin, bradykinin, PBu, A23187, and legends. In some experiments, cells were metabolically labeled with "Pi (36) prior to treatment with factors, lysis, and immunoprecipi- albumin-agarose were obtained from Sigma. VIC and thePKC peptide substrate [SerZ5]PKC-(19-31)were from Peninsula Laboratories, Inc. tation. Assays of Protein K i m e Activity in Anti-Tyr(P) Immunoprecipi- (Belmont, CA). Agarose-linked anti-Tyr(P) monoclonal antibody and tates-Immunoprecipitates prepared from 1.5 X 10' cells as described Raytide were purchased from Oncogene Science, Inc. The cyclic above were washed three times with lysis buffer and twice with 50 AMP-dependent protein kinase peptide substrate, Kemptide, the and the mM HEPES, pH7.4,O.l mM EDTA, 0.01% Brij, 75 mM NaCl (kinase casein kinase I1 peptide substrate ((Arg),-(Gl~)~-Thr-(Glu)~), assay buffer) and resuspended in 20 pl of this buffer. Kinase reactions peptide substrate based on the major autophosphorylation site were initiated by the addition of 10 mMMgC12 and 100 p M [-y-32P] (Tyr"') ofpp60""" were obtained from GIBCO-BRL. Py20 monoATP (20 pCi) and performed in atotal volume of 30 pl at 30 "C. After clonal anti-Tyr(P) antibody was from ICN. Carrier-free 32Pi(10 mCi/ anti-mouse IgG (15 incubation for 20 min (except where indicated otherwise), immuno- ml), [y3'P]ATP (5000 Ci/mmol), and 1251-sheep precipitates were washed twice with lysis buffer and analyzed by pCi/pg) were from Amersham (United Kingdom). All other reagents sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- used were of the purest grade available. PAGE) followed by autoradiography. In some experiments, phosphoRESULTS tyrosyl proteins were eluted from immunoprecipitates prior to SDSPAGE by incubation for 30 min a t 4 "C with 50 mM phenyl phosphate. NeuropeptideStimulation of Protein Kinase Activity in Labeling of protein bands was quantified by scanning the autoradiAnti-Tyr(P) Immunoprecipitates: Initial Rate and Effect of ograms of gels using an LKB Ultroscan densitometer. Orthouanadate-Intact, quiescent Swiss 3T3 cells were Phosphorylation of Tyrosine Kinase Peptide Substrates-Phosphorylation of both the broad specificity tyrosine kinase peptide treated with or without10 nM bombesin for 10 min and lysed, substrate, Raytide'" (Oncogene Science, Inc., Manhasset, NY), and a and anti-Tyr(P) immunoprecipitates were prepared. The repeptide substrate based on the tyrosine phosphorylation site (Tyr"') sulting immune complexes were incubated with [y3*P]ATP of pp60'"" (37, 38) was performed in kinase assay buffer as described for various times and then analyzed by SDS-PAGE. In imabove but in the presence of peptide substrate at concentrations of 150 and 500 p ~ respectively, , unless otherwise indicated. Peptide munoprecipitates prepared from bombesin-treated cells, we phosphorylation was stopped by the addition of 120 pl of 10% phos- observed a striking, time-dependentphosphorylation of a phoric acid, and the reaction mixture was then applied onto P-81 ion- major M , 115,000 band (p115), as well as phosphorylation of exchange chromatography paper. Papers were washed extensively several minor components, including twobands of M , 90,000 five times in 0.5% phosphoric acid, once with acetone, dried, and and 75,000 (Fig. lA).Control immunoprecipitates exhibited counted in ascintillation counter. a slow increase in the labeling of a band that exactly coRaytide is based on the sequence of human gastrin modified so that that it adheres to P-81 chromatography paper. The sequence of migratedwith p115. Densitometricscanningshowed labeling of the major p115 substrate in immune complexes human gastrin is pGlu-Gly-Pro-Trp-Leu-(Glu)5-Ala-Tyr-Gly-TrpMet-Asp-Phe-NHz. The pp60""" peptide substrate is based on the prepared from bombesin-treated cells increased linearly for amino acid sequence surrounding the major tyrosine autophospho- approximately 10 min after addition of [r-"P]ATP and thererylation site (residue T y P ) of pp60""": (Arg),-Leu-Ile-Glu-Asp-Ala- after continued to increase more slowly for a further 10 min Glu-Tyr-(Ala),-Arg-Gly-NH2(37,38). Phosphoamino Acid Analysis-This was performed according to (Fig. 1B). The initial rate of p115 phosphorylation was 14the method of Kamps and Sefton (39), as described. Briefly, after fold above the basal rate detected inimmunoprecipitates kinase assays and SDS-PAGE analysis of immunoprecipitates had derived from control, unstimulated cells (Fig. 1B). Identical been performed, 32P,-labeledprotein bands were transferred to Im- results t o those shown in Fig. 1 were obtained when immumobilon membranes according to the manufacturer'sinstructions noprecipitates were prepared using a different anti-Tyr(P) (Millipore Corp.), localized by autoradiography, and excised. Bands antibody (Py20). Inhibition of tyrosine phosphatases using were then subjected to hydrolysis in 5.7 M HCl for 1 h at 110 "C. Hydrolysates were lyophilized, resuspended in 10 pl of HzO/acetic orthovanadate added directly to immunoprecipitates at u p to acid/pyridine (1890100:10) and analyzed together with phosphoty- 500 pM slightly enhanced both basal and bombesin-stimulated rosine, phosphoserine, and phosphothreonine standards by electro- levels of p115 phosphorylation in anti-Tyr(P) immune comphoresis a t 650 V on microcrystalline cellulose thin layer plates. plexes (results not shown). Standards werelocalized using ninhydrin (0.2% in ethanol),and Similar analysis of anti-Tyr(P) immunoprecipitates preisotopically labeled phosphoamino acids were visualized by autora- pared fromcells treated witheithervasopressin, VIC, or diography. bradykinin also showed a marked increase in the labeling of Peptide Mapping-Peptide mapping of phosphotyrosyl proteins less was performed according to the method of Cleveland et al. (40). p115, although these peptide factors were consistently Briefly, 32Pi-labeledbands were localized by autoradiography, excised, effective in promoting kinase activity in immunoprecipitates and rehydrated for 30 min in 0.125 M Tris-HC1, pH 6.8, 0.1% SDS, 1 than bombesin (Fig. IC).In contrast, assaysof kinase activity

Neuropeptide Stimulation of Tyrosine Kinase Activity

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Phosphoamino Acid Analysis-Recently, we reported that bombesin, vasopressin, and endothelin/VIC rapidly increase tyrosinephosphorylation of bands of M , 115,000, 90,000, 81,000, and 75,000 in intact, quiescent Swiss 3T3 cells (36). 9 3The M , 115,000 band generated by neuropeptides in intact cells showed striking increases in serine, as well as tyrosine 69phosphorylation (36). Therefore, it was important to determine the specificity of the protein kinase activity detected in 5 20 1 5 10 20 anti-Tyr(P) immunoprecipitates preparedfrom neuropeptidelime, min stimulated Swiss 3T3 cells. Phosphoamino acid analysis revealed that bombesin, vasopressin, and VIC markedly inC D creased the phosphorylation of tyrosine residues of the p115 - r h o * PS band with increases of 8-, 6-, and 5-fold, respectively (Fig. 9 3I ID). In marked contrast to the results obtained with intact 6 9cells (36), the level of serine phosphorylation in immunoprePY cipitates was not alteredby neuropeptides and represented a - BOM V IVCP - BCM V I CV P BK negligible fraction (