Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Activation of protein kinase C (PKC) in quiescent. Swiss 3T3 cells using either the tumor ...
Vol. 268, No. 19, Issue of July 5, pp. 14261-14268.1993 Printed in U .S A .
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biolow. Inc.
Bombesin Stimulationof p125 Focal Adhesion Kinase Tyrosine Phosphorylation ROLE OF PROTEIN KINASE C, Ca’+ MOBILIZATION, AND THE ACTIN CYTOSKELETON* (Received for publication, February 25, 1993)
James Sinnett-Smith, Ian Zachary, Angela M. Valverde, and EnriqueRozengurtS From the Imperial Cancer Research Fund, P. 0. Box 123, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom
regeneration, and tumorigenesis (1). In particular, several Activation of protein kinase C (PKC) in quiescent Swiss 3T3cells using either the tumor promoter phor-neuropeptides, including bombesin, vasopressin, and endothebo1 12,13-dibutyrate(PDB) or diacylglycerolsinlin, are potent mitogens for quiescent Swiss 3T3 cells (1-3), creased the tyrosine phosphorylation of p125 focal a useful model system for the elucidation of signal transducadhesion kinase ( ~ 1 2 5by ~ 3.8-fold. ~ ~ ) PDB stimula- tion pathways leading to cell proliferation (4). These neurotion of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ was detected peptidesbind to specific high-affinity receptors (5-8) and within 1 min and reached a maximum within 5 min, stimulate a variety of early biochemical responses (2-4), inconsiderablyslower thanPDBstimulation of 80K/ cluding rapid hydrolysis of polyphosphoinositides, mobilizaMARCKS phosphorylation which was maximal within tion of intracellularCa2+, and activation of PKC’ (9-12). 1 min. In sharp contrast, bombesin-induced tyrosine Bombesin has also been shown to stimulate the release and phosphorylation of ~ 1 2 reached 5 ~ a~ maximum ~ (8fold stimulation) within 1 min after addition of the metabolism of arachidonic acid in Swiss 3T3 cells (13). The peptide and occurred with a half-maximal effect of receptors for bombesin, vasopressin, and endothelin belong 0.08 nM, 6-fold lower than the half-maximal effect of to the superfamily of receptors with seven hydrophobic putative transmembrane domains (14-18) and are coupled to bombesin on 80K/MARCKS phosphorylation. Downregulation of PKC by prolonged treatment with PDB effector molecules such as phospholipase C via GTP-binding 1 tyrosine % phos~ ~proteins ~ (19-22) of the Gq subfamily (23-25). blocked the effect of PDB on ~ Tyrosine phosphorylation has recently been implicated in phorylation but had no effect on the response to bombesin. A selectiveinhibitor of PKC, GF 109203X, the action of neuropeptide growth factors (26-33). Addition vasopressin, endothelin, and bradykinin to Swiss markedly inhibited the stimulation of ~ 1 2tyrosine 5 ~ ~of bombesin, ~ phosphorylation by PDB but had little effect on the 3T3 cells stimulates a rapid increase in tyrosine phosphorylresponseto bombesin,vasopressin, and endothelin. ation of a cluster of proteins of 110-130 kDa (28, 29). NeuBombesin stimulation of tyrosinephosphorylation ropeptide stimulation of Swiss 3T3 cells increases tyrosine could also be dissociated from mobilization of Ca2+ phosphorylation in cell-free preparations of these cells of both from intracellular stores. Depletion of the intracellular a major endogenous p115 substrates andof exogenously added Ca2+pool by treatment with the tumor promoter thappeptide tyrosine kinasesubstrates (30). Initial studiesshowed sigargin completely blocked the abilityof bombesin to that the substrates for neuropeptide-stimulated tyrosine phostransiently increase the cytosolic Ca2+ concentration phorylation were not related to known targets for receptor but had no effect on bombesin stimulation of ~ 1 2 tyrosine 5 ~ ~kinases ~ (reviewed in Ref. 34) such as the GTPasetyrosine phosphorylation. In contrast, cytochalasinD, activatingprotein,phospholipase C-7, or phosphatidylinositol an agent which selectively disrupts the network of 3”kinase (29). actin microfilaments, completely inhibited bombesinRecently, the novel cytosolic tyrosine kinasep125FAK, which and PDB-induced ~ 1 2 tyrosine 5 ~ phosphorylation. ~ ~ Within the same concentration range (0.3-2 KM), the was shown to localize to focal adhesion plaques (35, 36), has drug hadno effect on other early events stimulated by been identified as a prominent tyrosine-phosphorylated probombesin, including Ca2+ mobilization and activation tein in Swiss 3T3 cells stimulated by bombesin, vasopressin, and endothelin(31). Neuropeptide-induced ~ 1 2 phos5 ~ ~ ~ of PKC. These findings demonstrate that neither the PKC nor Ca2+ pathwaysare responsible for the rapid phorylation in Swiss 3T3 cells occurs at high stoichiometry and at concentrations of the peptides which parallel those stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ by neuropeptide growth factors. Furthermore, the integ- necessary for mitogenic stimulation (31). The striking rapidity rity of the actin cytoskeleton is essential for the effectsof neuropeptide-stimulated phosphorylation (detectable of both PDB and bombesin. within seconds) is consistent with p12!jFAKfunctioning as a downstream element in a neuropeptide-stimulated tyrosine kinase pathway. Several investigators have recently reported Neuropeptides stimulate DNA synthesis and proliferation in cultured cells and are implicated as growth factors in a variety of biological processes, including embryogenesis, tissue
* 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. 4 To whom all correspondence should be addressed. Tel.: 071-2693455; Fax: 071-269-3094.
~~
The abbreviations used are: PKC, protein kinase C; Anti-Tyr(P), anti-phosphotyrosine monoclonal antibody; BSA, bovine serum albumin; DiC8, 1,2-dioctanoyl-sn-glycerol;DMEM, Dulbecco’s modified Eagle’s medium; GRP, gastrin releasing peptide; IgG, immunoglobulin G; [Ca2+];, intracellular concentrationof Ca2+;methyl-TPA, 4-0-methyltetradecanoylphorbol 13-acetate; mAb, monoclonal antiPDB, phorbol12,13-dibutyrate; body; OAG, 1-oleoyl-2-acetylglycerol; ~ 1 2 5 ~p125 * ~ ,focal adhesion kinase; PAGE, polyacrylamide gel electrophoresis; 8OK/MARCKS, acidic 80-KDa/myristoylated alaninerich C kinase substrate.
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of ~ 1 2 5 Tyrosine ~ ~Phosphorylation "
that ~ 1 2 5 is" tyrosine-phosphorylated ~ ~ following activation of integrins as a consequence either of cell attachment to (36-39) or fibronectin and other extracellular matrix proteins of activation and aggregation of platelets (40). Furthermore,
buffer whose composition was given above. Lysates were clarified by centrifugation at 15,000 X g for 10 min and precleared by incubation with protein A-agarose for 1 h at 4 "C. After removal of protein Aagarose by brief (10 s) centrifugation, the supernatants were transferred to fresh tubes, and 80K/MARCKS was immunoprecipitated tyrosine phosphorylation of ~ 1 2 is 5elevated ~ ~ in ~chicken with 10 plof a rabbit polyclonal antibody (43) for 3 hat 4 "C. Protein embryo fibroblasts expressing activated variants of pp6Osrc A-agarose (50 pl) was then added tothe tubes for 1 h at 4 "C. and the oncogene productsof other avian sarcoma viruses (37, Immunoprecipitates were then washed five times with lysis buffer, 41). These findings suggest that tyrosine phosphorylation of resuspended in 2 X SDS sample buffer, and analyzed by SDS-PAGE. Down-regulation of PKC-PKC activity is extensively down-regu~ 1 2 5 constitutes " ~ ~ a point of convergence in the action of lated in Swiss 3T3 fibroblasts by prolonged pretreatment with PDB neuropeptides, integrins, and oncogenes (32). However, the (44-47). In the present studies, confluent and quiescent cultures were mechanism by which ~ 1 2 is5regulated ~ ~ by ~ extracellular pretreated with 800 nM PDB for 40 h in conditioned medium. In stimuli remains unknown. some experiments, pretreated cells were labeled with 32Pias described In the present paper, we examined whetherPKC activation, above also in the presence of 800 nM PDB. Conditioned medium is Ca2+mobilization, and cytoskeletal organization play a role taken from cultures of Swiss 3T3 cells grown in DMEM with 10% in mediating the stimulation of tyrosine phosphorylation by fetal bovine serum that have attained quiescence and is therefore depleted of growth-promoting activity. that direct bombesin and other neuropeptides.Wereport Measurement of [Ca'+/,-Confluent and quiescent cells in 90-mm activation of PKC by phorbol estersand membrane-permeant dishes were washed twice with DMEM and incubated for 10 min in diacylglycerols causes increased tyrosine phosphorylation of DMEM containing 1 p~ fura-2-tetraacetoxymethyl ester. After this ~ 1 2 5Surprisingly, ~ ~ ~ . however, several lines of evidence dem- time the cells were washed three times with electrolyte solution which contained 120 mM NaCl, 5 mMKC1, 1.8 mM CaCl,, 0.9 mM MgCI,, onstrate that bombesinstimulates ~ 1 2 tyrosine 5 ~ phos~ ~ phorylation through a mechanism that is independentof both 25 mM glucose, 16 mM Hepes, 6 mM Tris/HCl, and a mixture of acids at thesame concentrations as are presentin DMEM, pH PKC and the mobilization of intracellular Ca'+. In contrast, amino 7.2. The cells were then suspended in 2 ml of electrolyte solution by selective disruptionof actin filaments completely inhibits the gentle scraping and transferred to a 1-cm2quartz cuvette which was effects of bombesin andPDB o n ~ 1 2 tyrosine 5 ~ ~phosphoryl~ placed in a Perkin-Elmer LS-5 luminescence spectrophotometer. The ation, indicating that the maintenance of cytoskeletal integ- cell suspension was stirred continuously at 37 "C, and fluorescence was monitored at an excitation wavelength of 336 nm and emission rity plays an essential role in this signaling pathway. wavelength of510 nm. Factors were added to the cell suspension while fluorescence was being monitored as indicated. [Caz+Iiwas EXPERIMENTALPROCEDURES determined as described previously (10). Cell Culture-Stock cultures of Swiss 3T3 fibroblasts were mainMeasurement O ~ ' ~ ~ I - G Binding-Quiescent RP Swiss 3T3 cells were tained in DMEM supplemented with 10% fetal bovine serum in a washed twicewith DMEM and incubated with fresh DMEM containhumidified atmosphere containing 10% CO, and 90% air at 37 "C (6). ing 1mg/ml BSA and various concentrations of lZ5I-GRPas indicated, For experimental purposes, cells were plated either in 33-mm Nunc either in the absence or presence of 1.25 p~ cytochalasin D. At each Petri dishes at lo5 cells/dish, or in 90-mm dishes at 2.5 X lo5 cells/ concentration of radiolabeled ligand, binding was determined in the dish, inDMEM containing 10%fetal bovine serum and used after 6presence or absence of a 1000-fold excess of unlabeled bombesin. 8 days when the cells were confluent and quiescent (6). After 30 min, the cells were washed rapidly five times with ice-cold Immunoprecipitations-Quiescent cultures of cells werewashed phosphate-buffered saline containing 1 mg/ml BSA. Cells were solutwice with DMEM, treated with peptide factors in 1 ml of DMEM as bilized in 2% Na,CO,, 0.1 M NaOH, 0.1% SDS, and solubilizedcounts indicated, and lysed at 4 "C in 1 ml of a solution containing 10 mM were determined in a y counter (LKB). Nonspecific binding, deterTris/HCl, 5 mM EDTA, 50 mM NaCI, 30 mM sodium pyrophosphate, mined as cell-associated radioactivity not displaced by a 1000-fold 50 mM NaF, 100 p~ Na3V04,1% Triton X-100, and 1 mM phenyl- excess of unlabeled bombesin, was subtracted from the total binding methylsulfonyl fluoride, pH 7.6 (lysis buffer). Lysates were clarified to obtain specific binding. by centrifugation at 15,000 X g for 10 min and precleared by incubaMaterials-Bombesin, GRP, vasopressin, endothelin-8, PDB, tion with albumin-agarose for 1 h at 4 "C. After removal of albumin- DiC8,OAG, cytochalasin D, albumin-agarose, and agarose-linked agarose by brief (10 s) centrifugation, the supernatants were trans- anti-mouse IgG were obtained from Sigma. The specific PKC inhibferred to a fresh tube, and proteins were immunoprecipitated for 3 h itor, GF 109203X,was a gift from Dr. Kirilovsky at Laboratoires at 4 "C with agarose-coupled monoclonal antibodies either directed Glaxo,LesUlis, France. Thapsigargin and A23187were obtained against phosphotyrosine or ~ 1 2as5indicated. ~ ~ Immunoprecipitates ~ from Calbiochem-NovabiochemLtd., Nottingham, United Kingdom. were washed three times with lysis buffer and extracted for 10 min Agarose-linkedanti-Tyr(P) monoclonal antibody was purchased from at 95 "C in 2 X SDS-PAGE sample buffer (200 mM Tris/HCl, 6% Oncogene Science Inc., New York. Py20 monoclonal anti-Tyr(P) SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH 6.8) and antibody was from ICN, High Wycombe, United Kingdom, and the analyzed by SDS-PAGE and asdescribed under "Results" and in the 4G10 anti-Tyr(P) mAb was from UBI, Lake Placid, NY. The mAb figure legends. 2A7 directed against ~ 1 2 was 5 the ~ ~ generous ~ gift of Dr. Thomas Western Blotting-Treatment of quiescent cultures of cells with Parsons, University of Virginia. Carrier-free ["PIP; (10 mCi/ml), [ y factors, cell lysis, and immunoprecipitations were performed as de- "P]ATP (5000 Ci/mmol), lZ5I-GRP (2000 Ci/mmol), and lZ5I-sheep scribed above. After SDS-PAGE, proteins were transferred to Im- anti-mouse IgG (15 pCi/pg) were from Amersham (Amersham, United mobilon membranes (42). Membranes were blocked using 5% non- Kingdom). All other reagents used were of the purest grade available. fat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 3-5 h with either a mixture of the Py20 and 4G10 anti-Tyr(P) mAbs (1 pg/ml of each) or 2A7 a n t i - ~ l 2 5mAb ~~~ (1 pg/ml) as RESULTS indicated in phosphate-buffered saline containing 3% non-fat dried milk. Immunoreactive bands were visualized using '251-labeledsheep Activation of PKC by Phorbol Esters and Diacylglycerol anti-mouse IgG and autoradiography. Autoradiograms were scanned Stimulates ~ 1 2 5 ~Phosphorylation-To ''~ determine whether using an LKB Ultrascan XL densitometer and labeled bands quan- direct activation of PKC increased the tyrosine phosphoryltified using the Ultrascan XL internal digital integrator. The values are expressed as percentages of the maximum increase above the ation of p125FAK, quiescent Swiss 3T3 cells were treated for 10 min with 200 nM of PDB and lysed. Lysates were then control values. mAb, a n d the immunoprecipitates BOKIMARCKS Phosphorylation-Quiescent and confluent cells in incubated with anti-Tyr(P) 33-mm dishes were washed three times with phosphate-free DMEM were analyzed by Western blotting witha the n t i - ~ l 2 mAb 5~~~ and incubated with in 2 ml of phosphate-free DMEM containing 100 2A7. As shown in Fig. 1A (lanes 1 a n d 2), PDB caused a pCi/ml carrier-free ["'PIPi (1 Ci = 37 GBq) for 16 h to label the 5 ~ phosphorylation. ~ ~ Imendogenous ATP pool. The labeled cells were treated with factors as marked increase in ~ 1 2 tyrosine described, washedtwice with ice-cold Tris/saline solution (0.15 M munoprecipitation using mAb 2A7 followed by Western blotNaCl, 20 mM Tris/HCl, pH 7.5), and lysed at 4 "C in 100 pl of lysis ting with the same antibody (Fig. lA, lanes 3 and 4 ) showed
Bombesin Stimulation
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1
2 Tyrosine 5Phosphorylation ~ ~ ~
14263
Concentration Dependence and Time Course of Bombesin and PDB Stimulation of p12!jFAKTyrosine Phosphorylation-
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FIG.1. PDB and diacylglycerols stimulate~ 1 2tyrosine 5 ~ phosphorylation inSwiss 3T3 cells. A, quiescent Swiss 3T3 cells were incubated for 10 min at 37 “C with 200 nM PDB. Control cells (0) received an equivalent volume of solvent. Cells were then lysed and immunoprecipitates prepared using either anti-Tyr(P) (lanes 1 and 2) or mAb 2A7 (lanes3 and 4 ) . Immunoprecipitates were analyzed by SDS-PAGE followed by transfer of proteins to Immobilon (Millipore) and Western blotting withmAb 2A7. B and C, cells were treated for 10 min with various Concentrations either of PDB ( B ) or OAG (C).The cells were then lysed, and ~ 1 2 in5the~ clarified ~ ~ lysates was immunoprecipitatedwith mAb2A7; immunoprecipitates were analyzed by Western blotting with anti-Tyr(P) mAb. D, cells were treated with either 200 PM DiC8, 200 nM phorbol, or 200 nM methylTPA (MeTPA), andimmunoprecipitates were prepared from lysates of the cells using mAb 2A7 and analyzed by Western blotting with anti-Tyr(P). The position of ~ 1 2 is 5indicated ~ ~ ~ by an arrowhead. The positions of molecular weight markers ( X IO-’) are shown on the left. The results shown are representative of a t least three identical experiments. Other experimental details aredescribed under “Experimental Procedures.”
The kinetics of ~ 1 2 tyrosine 5 ~ ~phosphorylation ~ stimulated by either bombesin or PDB are shown in Fig. 2 (left). An increase in ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ was detected as early as 15 s after addition of bombesin to quiescentcells and reachedamaximum after 1 min. A salient feature of the results shown in Fig. 2 is that PDB-induced tyrosine phosphorylation was much slower comparedwith the effect of bombesin. Thus, an increase in ~ 1 2 tyrosine 5 ~ ~phosphoryl~ ation was only detectable after1 min and reached a maximum 5 min after additionof phorbol ester. Furthermore,bombesin consistently stimulateda higher level of phosphorylation even at saturating concentrations (200 nM)of PDB.Bombesin caused a 8.0 k 1.2-fold ( n = 17) stimulation compared with 3.8 f 0.6-fold ( n = 12) for PDB after 10 min of incubation. In sharp contrast to the very different time dependence displayed by bombesinand PDBin the stimulationof p12sFAK tyrosine phosphorylation, both factors stimulated the rapid ~phosphorylation ~ of theprominentPKCsubstrate80K/ MARCKS with very similar kinetics in parallel cultures of Swiss 3T3 cells. Maximum effects were obtained after 1 min of incubation with eitherbombesin or PDB. In addition, these agents were equally effective in stimulating 80K/MARCKS phosphorylationafter all times of treatment(resultsnot shown). Next, we compared theeffects of various concentrations of bombesin on ~ 1 2 and 5 80K/MARCKS ~ ~ ~ phosphorylation and Ca2+ mobilization in parallel sets of cultures. As shown in Fig. 2 (right), the half-maximal concentrations required for 80K/MARCKS phosphorylation and Ca2+mobilization were approximately 0.5 nM, 6-fold higher than that required
-
that recovery of ~ 1 2 from 5 ~ cell ~lysates ~ was not altered by I I II 2 40 treatment with PDB. An increase in tyrosine phosphorylation of ~ 1 2by5PDB ~ ~ ~ was also demonstrated when the lysates of cells were first immunoprecipitated with mAb 2A7 and the immunoprecipi0 2 4 6 8 10 0 0.01 0.1 1.0 10 tates Western-blotted with anti-Tyr(P) mAb (Fig. 1B). PDB Time, min Bombesin, nM enhanced ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ in a concentra2. Left, time course of bombesin and PDB stimulation of tion-dependent fashion; half-maximal effect was elicitedat 10 ~ FIG. 1 2 tyrosine 5 ~ phosphorylation. ~ ~ Cultures of Swiss 3T3 cells were nM and a maximum effect at 50 nM (Fig. 1B). Whereas the treated for various times as indicated with either 10 nM bombesin mAB 2A7 was raised against the chicken p125FAK,identical (closed circles) or 200 nM PDB (open circles) and lysed. Tyrosine experiments using a polyclonal antibody against the C-terphosphorylation ofp12fjFAK was analyzed by immunoprecipitation using mAb 2A7 and Western blotting with anti-Tyr(P). Quantificaminal portion of the murine ~ 1 2 (36) 5 also ~ ~demonstrated ~ 5 ~ ~ ~ was performed by scanning densistimulation of ~ 1 2 tyrosine 5 ~ ~phosphorylation ~ inSwiss 3T3 tion of ~ 1 2 phosphorylation tometry, and thevalues shown are themeans f S.E.( n= 5 ) expressed cells treated with PDB (results not shown). as percentages of the maximum increases above the control unstimTreatment of intact cells with the membrane-permeant ulated values. Right, concentration dependence for bombesin stimudiacylglycerol OAG, an analogue of the endogenous activator lation of ~ 1 2 tyrosine 5 ~ phosphorylation, ~ ~ Ca2+ mobilization, and of PKC (44), similarlycaused a concentration-dependent 8OK/MARCKS phosphorylation 3T3 cells. Quiescent cells were treated with various concentrations of bombesin, lysed, and used for increase in ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ (Fig. 1C). In 5 ~ phosphorylation ~ ~ by immunoprecipiaddition, thediacylglycerol, DiC8, also increased the tyrosine evaluation of ~ 1 2 tyrosine tation with mAb 2A7 and anti-Tyr(P) Western blotting (closed cirphosphorylation of ~ 1 2 in 5intact ~ ~ Swiss ~ 3T3 cells, whereas cles). Parallel cultures were preloaded with fura-2 and transferred to the biologically inactive phorbol ester methyl-TPA or phorbola cuvette prior to treatmentwith bombesin; these cells were used for both a t 200 nM had noeffect on ~ 1 2 tyrosine 5 ~ phosphoryl~ ~ measurement of [Ca*+li(open sqwres) as described under “Experiation (Fig. 1D). mental Procedures.” Parallel cultures of cells were labeled with [”PI The results presented inFig. 1suggested that PKCactiva- Pi, treated with bombesin a t the concentrations indicated, and used tion could provide a potential mechanismby which bombesin for measurement of 80K/MARCKS phosphorylation (closed squares) as described under“Experimental Procedures.” Quantification of and other neuropeptides stimulate tyrosine phosphorylation phosphorylation was performed by scanning densitometry. Values of ~ 1 2 5 Consequently ~ ~ ~ . the following experiments were shown are the mean 9 S.E. of three independent experiments and designed to examine in detail the role of PKC in bombesin- are expressed as percentages of the maximum increase above control induced ~ 1 2 tyrosine 5 ~ ~ phosphorylation. ~ unstimulated values. ‘ I
Bombesin Stimulation
14264
of ~
1 2 Tyrosine 5Phosphorylation ~ ~ ~
for ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ (Fig. 2). At a concentration of0.1nM, ~ 1 2 . phosphorylation 5 ~ ~ ~ was 60% of the response achieved at maximum concentrationsof the peptide, compared with only 10 and 18% for bombesin stimulation of Ca2+mobilization and 80K/MARCKS phosphorylation, respectively. The results shown in Fig. 2 indicate that bombesin stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ precedes the increase in phosphorylation which occurs in response to direct activators of PKC and occurs at a lower level of receptor occupancy than that required bombesin for activation of PKC. These observations suggest that PKC activationcould not be entirely responsible for mediating the stimulationof ~ 1 2 tyrosine phosphorylation by bombesin. PKC Down-regulation or InhibitionDoes Not PreventBom5 ~ ~ Phosphorylation-Down~ besin-Stimuluted ~ 1 2 Tyrosine regulation of PKC by prolonged exposure of cells to PDB has been extensively used to dissect the role of this pathway in the mitogenic response tobombesin and other neuropeptides. Thus, chronic pretreatment with PDB blocks bombesin stimulation of 80K/MARCKS phosphorylation and eitherblocks or attenuates other early and later responses mediated by bombesin activation of PKC (8).Consequently, we examined the effect of pretreating cells with 800 nM PDB for 40 h on the stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ caused by a subsequent addition of PDB andbombesin. Anti-Tyr(P) immunoblotting of mAb 2A7 immunoprecipitates showed that stimulation of p12SFAK tyrosine phosphorylationeven by maximal concentrationsof phorbol esterwas markedly reduced in cells which had previously been chronically exposed to PDB (Fig. 3). Inthreeindependentexperiments,PDB-induced ~ 1 2 tyrosine 5 ~ ~phosphorylation ~ was reduced by 82% in PKC down-regulated cells. In contrast, down-regulation of PKC had no substantialeffect on bombesin stimulation of p12sFAK tyrosine phosphorylation either a t 10 nM or at 0.1 nM (Fig. 3). Inparallelcultures of cells, down-regulation of PKC blocked the effect of both bombesin andPDBon80K/ MARCKS phosphorylationover the same range of concentrations used todetermine ~ 1 2 . tyrosine 5 ~ ~ phosphorylation ~ (results not shown). Inhibitors of PKC have been widely used to examine the role of this kinase in signaling pathways.Recently, the staurosporine-related compound,GF 109203X, was reported tobe a highly selective inhibitor of PKC in Swiss 3T3 cells a t
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FIG.3. Effect of PKC down-regulation on PDB and bombesin-stimulated p12EiPAK tyrosine phosphorylation. Quiescent Swiss 3T3 cells were pretreated either in the absence (open burs) or presence (closed burs) of 800 nM PDB for 40 h and then challenged for 10 min with either bombesin at 10 or 0.1 nM or with 200 nM PDB. Controls received an equivalent volume of solvent. Cells were then lysed, and p12fjFAKwas immunoprecipitatedusing mAb 2A7. Immunoprecipitates were subsequently analyzed by Western blotting with anti-Tyr(P).Values shown are the mean & S.E. of three independent determinations and areexpressed as the percentage of the maximum increase above control unstimulated levels. The maximum increase is that elicited by 10 nM bombesin in unpretreated cells.
concentrations which had no effect either on CAMP-dependent kinase or on the platelet-derived growth factor, epidermal growth factor,andinsulin receptor tyrosinekinases (48). These findings suggested that GF 109203X would be useful for examining the involvement of PKC in the stimulation of ~ 1 2 tyrosine 5 ~ ~phosphorylation. ~ Pretreatment of Swiss 3T3 cells with 3.5 I . ~ MGF 109203X for 1 h blocked bombesin- and PDB-induced 80K/MARCKS phosphorylation (Fig. 4 ( A ) ) . Inparallelcultures,GF 109203X markedlyreduced PDB stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation. ~ ~ In contrast, theinhibitorhadlittle effect on bombesin stimulation of ~ 1 2 tyrosine 5 ~ ~ phosphorylation ~ (Fig. 4A). The effect of GF 5109203X ~ ~ on ~ PDB-induced ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ was concentration-dependent with half-maximal and maximum effects of 2.5 and 5 PM, respectively (Fig.4B). Over the same concentration range the inhibitor had little effect on bombesin stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ (Fig. 4B). We recently reported that in addition to bombesin, vasopressin and endothelin also stimulate the rapid tyrosine phosphorylation of p12sFAK in Swiss 3T3 cells (31).These peptides also cause a rapid activation of PKC, as judged by BOK/ MARCKS phosphorylation (Ref. 49 and Fig. 4). To further investigate the role of PKC in mediating vasopressin and endothelin stimulationof tyrosine phosphorylation, theeffect of GF109203X was examined.In accordwith theresults obtained for bombesin,the inhibitor, aatconcentration which blocked the response to PDB (3.5 PM), had little effect on the increase in~ 1 2 tyrosine 5 ~ ~ phosphorylation ~ caused byeither vasopressin or endothelin, but markedly reduced the stimulation of 80K/MARCKS induced by these peptides (Fig. 4C). Dissociation of ~ 1 2 Tyrosine 5 ~ ~Phosphorylation ~ from Ca2+ Mobilization-The results shown above indicated that activation of PKC could notaccount for thestimulation of tyrosine phosphorylation of ~ 1 2 by5mitogenic ~ ~ ~neuropeptides. Activation of phospholipase C by bombesin, vasopressin, and endothelin promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to production of inositol 1,4,5-trisphosphate, which in turn mediates the rapid mobilization of Ca2+from intracellular stores (2-4). It was plausible, therefore, that an increase cytosolic in Ca2+could explain the effects of regulatory peptidesonthistyrosinekinase pathway. Treatment of Swiss 3T3 cells either for 1 or 10 min with the Ca2+ ionophore, A23187, a t a concentration of 100 nM, which induced an increase in [Ca2+];comparable with that 5~~~ induced by bombesin(Fig. 5 (left)), i n ~ r e a s e d p 1 2 tyrosine phosphorylation to only 10% of the maximum phosphorylation elicited by bombesin (Fig. 5 (right)). We next examined the effect of the tumor promoter thapsigargin. This agent specifically inhibits the endoplasmic reticulum Ca*+-ATPase and thereby depletes Ca2+ from intracellular compartments (50). Treatment with 30 nM thapsigargin for 30 min blocked the increase in [Ca2+]; induced by subsequently added bombesin(Fig. 5, (left))buthadno effect ontheincreasein p12sFAK tyrosine phosphorylation in parallel cultures caused by either 1- or 10-min exposure to 10 nM bombesin (Fig. 5 (right)).The increase in cytosolic Ca2+caused by treatment with thapsigargin was not accompanied by anydetectable increase intyrosinephosphorylation of ~ 1 2 (Fig. 5 ~5 ~ ~ (right)). Cytochalasin D Blocks Bombesin Stimulation of ~ 1 2 5 ~ ~ Tyrosine Phosphorylation-Given the localization of ~ 1 2 5 ~ ~ in focal adhesions which form at the termini of actin stress fibers (51), we reasoned that the integrity of the cytoskeleton, and particularly the actin filamentnetwork, could be neces-
Bombesin Stimulationof ~ 1 2 Tyrosine 5 ~ ~Phosphorylation ~ PDB
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GF 109203X, pM FIG.4. Effect of a selective PKC inhibitor on ~ 1 2 tyrosine 5 ~ phosphorylation. ~ ~ A, quiescent Swiss 3T3 cells were pretreated for 1 h either in the absence(-) or presence(+) of GF109203X. Control cells received an equivalent volume of solvent. Cells were then incubated for a further 10 min either in the absence ( C o n t ) or in the presence of 10 nM bombesin (Born) or 200 nM PDB. Cells were then lysed, and immunoprecipitates of ~ 1 2 were 5 ~analyzed ~ ~ by anti-Tyr(P) Western blotting. Parallel cultures of cells were labeled with ["'PIPi and subsequently treated with GF109203X and then eitherbombesin or PDB. These[:'"P]Pi-labeled cells were then used to measure phosphorylation of 80K/MARCKS. B, cells were pretreated for 1 h with the concentrations of GF109203X indicated and subsequentlychallenged for 10 5 ~ ~ ~ min with either 10 nM bombesin (closed circles) or 200 nM PDB (open circles). The cells were then lysed, and ~ 1 2 immunoprecipitates were prepared and analyzed by anti-Tyr(P) Western blotting as described under "Experimental Procedures." Quantification of ~ 1 2 5 ~ ~ tyrosine phosphorylation was determined by scanning densitometry of autoradiograms. The values from two independent experiments are expressed as percentages of the maximum increase in~ 1 2 5 tyrosine ' ~ ~ phosphorylation above control, induced by either PDB or bombesin. C , cells were pretreated for 1 h either in the absence (-) or presence (+) of GF 109203X. Control cells received an equivalent volume of solvent. Cells were then incubated for a further 10 min either in the absence( C o n t ) or in the presence of 20 nM vasopressin ( V P ) or 50 nM endothelin ( V I C ) . Cells were lysed and immunoprecipitates of p125 FAK were analyzed by anti-Tyr(P) Western blotting. Parallel cultures were labeled with ["'PIP, and subsequently treated with either vasopressin or endothelin. These ["P]Pi-labeled cells were then used to measure phosphorylation of 80K/MARCKS.
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1 10 30
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-
30 1
1
"
-
30
10 10
A231 07 Thapsigargin Bornbesin
Time, min FIG. 5. Effect of A23187 and thapsigargin on bombesin stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ in Swiss 3T3cells. Left, quiescent cultures of cells were incubated with fura-2-tetraacetoxymethyl ester and transferred to a cuvette by gentle scraping. Some cells were pretreated for 30 min with 30 nM thapsigargin prior to loading with the Ca2+indicator. [Ca'']; was continuously measured in a luminescence spectrometer as described under "Experimental Procedures." The basal [Ca'+Ii was monitored for 1 min before treating the Bombesin a t 10 nM was also added to cells which had cells either with 100 nM A23187, 10 nM bombesin (Born),or 30 nM thapsigargin (TG). been pretreated for 30 min with 30 nM thapsigargin. Right, parallel cultures of cells to those used for measurement of [Ca2+Ii shownin the left panel were treated for the times shown with 100 nM A23187, 30 nM thapsigargin, or with 10nM bombesin as indicated. Some cells were pretreated with 30 nM thapsigargin for 30 min (cross-hatched bars)and subsequently treatedwith 10 nM bombesin for either 1or 10min. As indicated thecells were then lysed, and ~ 1 2 immunoprecipitates 5 ~ ~ ~ were prepared andanalyzed by anti-Tyr(P) Western blotting as described previously. Quantification of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ was determined by scanning densitometry of autoradiograms. The values 5 ~ phosphorylation ~ ~ above control shown are the means f S.E. ( n = 3) expressed as precentages of the maximum increase in ~ 1 2 tyrosine unstimulated levels.
Bombesin Stimulation
14266
of ~
1 2 Tyrosine 5 ~ ~Phosphorylation ~
sary for the stimulationof ~ 1 2 tyrosine 5 ~ phosphorylation. ~ ~ membrane-permeable diacylglycerols stimulates tyrosine Consequently we examined whether disruption of the actin phosphorylation of the novel cytosolic tyrosine kinase cytoskeleton could interferewiththeincreasein ~ 1 2 5~ 1~ 2 ~5 The ~~ concentration ~ ~ . dependence of PDB-induced tyrosine phosphorylation induced by bombesin. ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ closely paralleled the conTo test this possibility, quiescentSwiss 3T3 cells were centration dependencefor the effect of phorbol esters on both pretreated for 2 h with different concentrations of cytochal- PKC activation, as judged by 80K/MARCKS phosphorylaasin D, an agent which selectively disrupts the network of tion, and on mitogenesis. Furthermore, the stimulation of actin filaments (52), and then challenged with bombesin for ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ induced by PDB or OAG a further 10 min. The cells were then lysed and ~ 1 2 was 5 ~was ~ markedly ~ decreased either by down-regulation or inhibiimmunoprecipitated and analyzed by anti-Tyr(P) Western tion of PKC. These results indicate that activationof PKC blotting. As shown inFig. 6 ( A and B ) ,cytochalasin D blocked in intact cells is a potential signaling pathway leading to bombesin stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ in enhanced tyrosine phosphorylation of ~ 1 2 5 ~ ~ ~ . a concentration-dependent manner; half-maximal effect was Tyrosinephosphorylation of ~ 1 2 has 5 recently ~ ~ ~ been obtained a t a concentration of 0.6 ~ L Mand maximum effect identified as a novel early eventin the actionof neuropeptide was achieved a t 1.2 p ~ The . drug also inhibited PDB-induced growth factors in Swiss 3T3 cells (reviewed in Ref. 32). The tyrosine phosphorylation of ~ 1 2 5Immunofluorescence ~ ~ ~ . of molecular mechanisms by which neuropeptide receptor actiparallel cultures of cells which had been fixed and stained vation leads to anextremely rapid increase in~ 1 2tyrosine 5 ~ ~ ~ with tetramethylrhodamine B isothiocyanate (TR1TC)-con- phosphorylation are poorly understood. Bombesin and other jugated phalloidin showed that cytochalasin D over the same neuropeptides are known to induce the rapid hydrolysis of range of concentrations profoundly disrupted both the netinositolphospholipids to generate the intracellular second work of actin microfilaments in quiescent Swiss 3T3 cells and messengers diacylglycerol and inositol 1,4,5-trisphosphate inbombesin-treated cells (resultsnotshown).Inmarked contrast to theeffect of cytochalasin D, the anti-microtubule which activate PKC andmobilize Ca”, respectively. Since, as agent colchicine up to a concentration of2.5 p~ did not shown in the present study, direct PKC activation leads to ~ 1 2 phosphorylation, 5 ~ ~ ~ it was logical to assume that PKC reduce the stimulation of ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ could mediate entirely or partly neuropeptide stimulationof in response to either bombesin or PDB(Fig. 6 (C)). 5 ~ phosphorylation. ~ ~ Surprisingly, several lines It was important to establish that the effect of cytochalasin ~ 1 2 tyrosine of evidence indicate that this is not the case. 1) BombesinD on ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ by bombesinwas induced phosphorylation of ~ 1 2 is5consistently ~ ~ ~ greater specific. Pretreatment with the drug had no effect either on the affinity or numberof receptors for bombesin as shown by than the maximum effect elicited by either PDB or OAG; 2) 5 ~ phosphoryl~ ~ Scatchard analysisof binding measurements of the mamma- strikingly, bombesin stimulates ~ 1 2 tyrosine bombesin and PDB lian bombesin-related peptide, ‘*“I-GRP, to intact Swiss 3T3 ation more rapidlythan PDB; in contrast, used stimulated anidentical rapid PKCcells (Fig. 7 (upper)).Furthermore, cytochalasin Ddid not at the concentrations inhibit bombesin stimulation of either 80K/MARCKS phos- mediatedphosphorylation of 80K/MARCKS;3) bombesin stimulates ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ at concentraphorylation or the mobilization of Ca” fromintracellular stores (Fig. 7 ) . tions considerably lower than those required to activate PKC as judged by phosphorylation of 80K/MARCKS; 4) downDISCUSSION regulation of PKC by chronic pretreatmentof cells with PDB effect of PDB but has no substantial In the present paperwe demonstrate that direct activation abrogates the subsequent of PKC either by biologically active phorbol esters or by effect on theresponse to eitherlow or high concentrations of A Cytochalasin D, pM 0
0.15 0.3
B
-
0.6 1.25 2.5
PDB
+
c
Cont COlC.
-
+
-
Born
+
0 ”
PDB
-
+
i‘\
100
Or
80
E
3
60
0
-
-
+
40
Cytochalasin D, pM
FIG.6. Effect of cytochalasin D and colchicine on bombesin stimulation of p12€iFAK tyrosine phosphorylation. A , quiescent Swiss 3T3 cells were treated for 2 h in the presence of various concentrations of cytochalasin D as indicated and then incubated in the presence of 10 nM bombesin for a further 10 min. Parallel cultures were incubated for 2 h either in the absence (-1 or presence (+) of 1.25 P M cytochalasin Dand subsequently treated for afurther 10 min with 200 nM PDB. The cells were then lysed, ~ 1 2was 5 immunoprecipitated ~ ~ ~ from the lysates using mAb 2A7,and theimmunoprecipitates were analyzed by anti-Tyr(P)Western blottingas described under “Experimental ~ ” ~ after pretreatment with various concentrations of Procedures.” B, the bombesin-induced anti-Tyr(P) immunoreactivity of ~ 1 2 5 obtained cytochalasin D (shown in A ) was quantified by scanning densitometry (closed circles). Control cells incubated in the absence of bombesin but with various concentrations of cytochalasin D (omitted from A for clarity) are also presented in this panel (open circles). In this experiment, the values shown represent percentages of the maximum phosphorylation which was induced by 10 nM bombesin in unpretreated cells. C, quiescent cells were incubated for 2 h in the absence (-) or in the presence (+) of 2.5 PM colchicine and then treated for a further 10 min either with 10 nM bombesin or with 200 nM PDB. Control cells were incubated with an equivalent volume of solvent. The cells were then lysed, and anti-Tyr(P) immunoreactivity of ~ 1 2 was 5 determined ~ ~ ~ as described under “Experimental Procedures.”
Bombesin Stimulationof ~ 1 2 Tyrosine 5 ~ ~Phosphorylation ~
I
14267
has also been reported that the induction of Ca'' oscillations are required for GPIIbIIIa-induced tyrosine phosphorylation of a 125-kDa protein incells expressing this integrin(54). It is likely but unproven that these 125-kDa bands are related 0.06 to ~ 1 2 5In~view ~ ~of .these findings and the rapid kinetics of neuropeptide-stimulated Ca" mobilization and ~ 1 2 ty5 ~ ~ ~ 0.03 rosinephosphorylation, we testedwhether Ca'+ could be responsible for the effects of neuropeptides on tyrosine phosphorylation. We found that neither Ca'+ ionophore nor thapsigargin caused a Ca'+-dependent increase in ~ 1 2 tyrosine 5 ~ ~ ~ 1251-GRP Bound (pmoles/106 ) phosphorylation in Swiss 3T3 cells. Furthermore, depletion of the intracellular Ca2+ pool by treating cells with thapsigargin blocked the mobilization of Ca" by a subsequent addition Cont PDB Bom of bombesin, but had no effect on ~ 1 2 tyrosine 5 ~ phos~ ~ - + - + - + CD phorylation. In addition, bombesin stimulated ~ 1 2 tyro5 ~ ~ ~ 80KI sine phosphorylation with a half-maximal concentration 6MARCKS fold lower than that requiredfor mobilization of Ca'+. Thus, several lines of evidence indicate that increases in [Ca'+Ii do not mediate neuropeptide-stimulated ~ 1 2 tyrosine 5 ~ ~ phos~ 1 min phorylation. + CD The findings presented here indicate that the two major 800 800 early signals generated by activation of phospholipase C are not responsible for neuropeptide stimulation of ~ 1 2 ty-5 ~ ~ ~ rosine phosphorylationin Swiss 3T3 cells. This novel protein tyrosine kinase has attracted interestbecause of its subcellu200 200 - 4 lar localization to focal adhesions (35, 36). Focal adhesions, Bom,lOnM Bom,lOnM which form at the terminiof actin stress fibers, are thought FIG.7. Effect of cytochalasin D on 12'I-GRP binding and to playacrucial role in the processeswhichregulate cell bombesin stimulation of SOK/MARCKS phosphorylation and adhesion and motility. Integrins cluster a t these sites followCa2+mobilization. A , Scatchard analysis of"'1-GRP binding in ing attachment of cells to the extracellular matrix (51) and control and cytochalasin D-treated cells. Quiescent Swiss 3T3 cells have been shown to promote ~ 1 2 tyrosine 5 ~ phosphoryla~ ~ were washed twice with DMEM and incubated for 2 h in 2 ml of tion (35-40). The molecular basis of focal adhesion and stress DMEM either in the absence (open circles) or in the presence(closed circles) of 1.25 p M cytochalasin D. The medium was then removed, fiber formation remains unknown. Several recent reports have implicated the rho p21 family, and the cells were incubated with fresh DMEM containing 1 mg/ml BSA and various concentrations of 12sI-GRPas indicated, either in which belongs to the ras p21-related small G protein supertheabsence(open circles) orpresence (closed circles) of 1.25 p~ family (55), in theassembly of focal adhesion plaques and in cytochalasin D. After 30 min, thecells were washed rapidlyfive times the regulation of the actin cytoskeleton in Swiss 3T3 cells with ice-cold phosphate-bufferedsalinecontaining 1 mg/ml BSA. The cells were then solubilized, and specific binding of T - G R P was (56, 57). Microinjection of rho p21 into these cells increases of either GDP determined as described under "Experimental Procedures." The val- the actinfibers (56). Conversely, microinjection ues shown represent the mean of triplicate determinations which dissociation inhibitor, which is thought to increase the GDPagreed within 10%. B , bombesin and PDB stimulate 80K/MARKS bound (inactive) stateof rho p21, or of the ADP ribosyltransphosphorylation in control and cytochalasin-treated cells. Quiescent ferase Cn, which impairs rho p21 function, causes the disrupcultures were labeled with ["P]Pi for 4 h. During the last 2 h of the tion of the actin filament network (57). Bombesin has been labeling period, cells were also incubated either with cytochalasin D shown to promote a rapid increase in stress fibers and focal ( C D )a t 1.25 p~ (+) or with an equivalent volume of solvent (-). The cells were then treated for 10 min with either 10 nM bombesin or 200 adhesions, an effect apparently mediatedby rho p21 (56). In the present study we demonstrate thatbombesin-induced nM PDB,and80K/MARKSphosphorylationwasdeterminedas described under "Experimental Procedures." C, bombesin increases ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ is completely blocked by [Ca2+Ii in control and cytochalasin D-treated cells. Quiescent cells treatment with cytochalasinD, which disrupts the actinfilawere incubated for 2 h either in the absence or presence (+CD) of ment network. In contrast, dissolution of microtubules ex1.25 p M cytochalasin D and then incubated with fura-2-tetraacetoxerted no inhibitory effect on ~ 1 2 tyrosine 5 ~ phosphoryla~ ~ ymethyl ester. The effect of bombesin on [Ca2+Iiwas determined as tion. Thus, it is attractive to speculate that ~ 1 2 tyrosine 5 ~ ~ ~ described under "Experimental Procedures." phosphorylation induced by bombesinis triggered by a change bombesin; 5) a highly selective inhibitor of PKC blocked the in microfilament organization which, in turn, is mediated by increase in~ 1 2tyrosine 5 ~ ~ phosphorylation ~ induced by PDB rho p21 activation. However, we cannot eliminate an alternative interpretation, namely, that ~ 1 2 tyrosine 5 ~ phos~ ~ but did not impair theresponse to bombesin. This is consistphorylation requires the integrity of the microfilament netent with our previous findings that desensitization of the work which would play a permissive rather than signaling PKC pathway blocked PDB stimulation of phsophorylation role. Indeed, ~ 1 2 tyrosine 5 ~ phosphorylation ~ ~ inresponse to in anti-Tyr(P) immunoprecipitates both of an endogenous bombesin (Fig. 2) appears to precede any detectable change p115 band and exogenously added peptide substrate, but did in actin filament organization (56). Furthermore, Swiss 3T3 not impair the response to bombesin (30). Taken together, cells brought to quiescent in the presence of serum (asin this these results provide compelling evidence that neuropeptides study)retain a well developed microfilament network' in stimulate tyrosine phosphorylationof ~ 1 2 through 5 ~ a~sig-~ contrast to serum-starved cells used in experiments in which nal transduction pathway that is largely independent of PKC. bombesin was shown to induce focal adhesion assembly and In liver epithelial cells, angiotensin I1 increases tyrosine phosphorylation of cellular components, includinga 125-kDa J. Sinnett-Smith, I. Zachary, A. M. Valverde, and E. Rozengurt, band, apparently through a Ca2+-dependent pathway (53). It unpublished results. 0.09
-
-A -A -+
Bombesin Stimulation of ~ 1 2 5:A' K Tyrosine Phosphorylation
14268
A. V., Hepler, J. R., Brown, K. 0..and Sternweis, P. C. (1991) actin fibers (56). Although further experimental work will be 23. Smrcka, Scrence 251,804-807 necessary to elucidate whether p12EiFAK,rho p21, and actin 24. Taylor, S., Chae, H., Rhee, S. G., and Exton, J. (1991) Nature 3 5 0 , 5166151 filaments lie on a linear signal transduction pathway, it is 25. W;:k., Lee, C. H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. clear that the maintenanceof the actincytoskeleton is essen2 6 7 , 1811-1817 26. Huckle, W. R., Prokop, C. A., Dy, R. C., Herman, B., and Earp, S. (1990) tial for the stimulationof p12tjFAK tyrosine phsophorylation. Mol. Cell. Biol. 10, 6290-6298 In conclusion, our resultsshow that p12ijFAK tyrosine phos- 27. Force, T., Kyriakis, J. M., Avmch, J., and Bonventre, J. V. (1991) J. Biol. Chem. 266,6650-6656 phorylation can be induced by direct activation of PKC in 28. Leeb-Lundberg, L. M. F., and Song, X.-H. (1991) J. Biol. Chem. 2 6 6 , intact cells. Surprisingly, bombesin stimulates ~ 1 2 tyro5 ~ ~ 7746-7749 ~ I., Gil, J., Lehmann, W., Sinnett-Smith, J. and Rozengurt, E. sine phosphorylation through a PKC-independent pathway. 29. Zachary, (1991) Proc. Natl. Acad. Sei. U. S. A. 88, 4577-4581' The integrity of the polymerized actin network is essential 30. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1991) J. Biol. Chem. 266,24126-24133 for the function of both PKC-dependent and independent 31. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1992) J. Biol. Chem. pathways of ~ 1 2 tyrosine 5 ~ phosphorylation. ~ ~ 2 6 7 , 19031-19034 Acknowledgment-We are indebted to Dr. T . Parsons (University of Virginia) for the generous supply of mAb 2A7.
1. 2. 3. 4. 5. 6.
7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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