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Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin‐related peptide, gastrin‐releasing peptide.

Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the... The EMBO Journal vol.5 no. 11 -2898, pp.2889 1986 Early events phosphorylation the treatment of Swiss following 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide Clare M.Isacke1, Jill tion Meisenhelder', Kenneth of D.Brown2, phosphatidylinositol (PI) breakdown, which a generates Kathleen L.Gould13, Stephen J.Gould3 and Tony transient Hunter' intermediate, that binds to and diacylglycerol (DAG), activates kinase C in protein (reviewed Nishizuka, 1984; 'Molecular Biology and Virology Laboratory, The Salk Institute, PO Box Protein kinase C also acts as a cellular 85800, San Diego, Ashendel, 1985). major CA 92138, USA, 2AFRC Institute of Animal Physiology, for tumor such as Babraham, Cambridge CB2 4AT, UK and receptor promoters 3Department of 12-O-tetradecanoyl-phor- Biology, University of California at San Diego, La Jolla, CA 92093, USA bol-13-acetate and (TPA) phorbol dibutyrate which can (PDBu), substitute for DAG in the Communicated activation of the by R.Dulbecco enzyme. Thus, activ- ation of kinase C is protein to an essential role in likely play the Bombesin and the related mammalian peptides, such as stimulation elicited mitogenic tumor by promoters (reviewed in gastrin-releasing peptide (GRP), are potent mitogens for some Nishizuka, 1984; Ashendel, 1985). fibroblast cell lines. Here we have examined the bombesin- Bombesin is a tetradecapeptide isolated from the skin of Bom- and GRP-mediated changes in the phosphorylation of pro- bina bombina et Several (Anastasi al., 1971). peptides structurally teins in Swiss 3T3 cells and compared these to the events related to bombesin have been isolated from mammalian tissue observed after platelet-derived growth factor (PDG1F), epider- and are they characterized a N by having pyroglutamyl terminus mal growth factor (EGF) and tumor promoter treatment. In and a highly homologous C-terminal octapeptide. The best agreement with previous reports, bombesin, GRP and PDGF, characterized is gastrin-releasing peptide (GRP; McDonald et but not al., EGF, increased the activity of protein kinase C. This which 1979), with bombesin for to competes binding high-affinity was assayed by an inhibition of binding, stimula- [1251]EGF cell surface receptors on cells target (Westendorf and Schon- tion in phosphorylation of pp6oc-ws on serine 12 and stimula- brunn, and and elicits 1983; Zachary Rozengurt, 1985) identical tion in phosphorylation of a group of 80 kd proteins. The when responses injected into experimental animals (Brown et al., different phosphorylated forms of the 80 kd proteins were The 1980). bombesin-related peptides have a wide range of examined by tryptic peptide mapping and shown to contain physiological effects including the stimulation of hormone release multiple phosphorylation sites. An investigation of the tyrosine in vivo and in vitro (e.g. Rivier et al., 1978; Westendorf and phosphorylation events following mitogen treatment reveal- Schonbrunn, and 1982; Swope Schonbrunn, 1984) and stimula- ed a significant difference between PDGF and the bombesin tion of cell division in cultured fibroblasts (Rozengurt and Sinnett- peptides. PDGF treatment caused a marked increase in total Smith, 1983) and human bronchial epithelial cells (Willey et al., cellular phosphoyrosine levels, and trosine phosphorylation 1984). both of known substrates and its own receptor. In contrast, it Recently has been that suggested bombesin-related peptides bombesin and GRP treatments resulted in only a weak or are involved in the autocrine stimulation of human small-cell lung undetectable increase in tyrosine phosphorylation of total carcinomas as these cells respond mitogenically to and express cellular protein or known substrates. In this respect bombesin high-affinity receptors for bombesin-related peptides (Weber et and GRP were more similar to EGF. The fact that the al., 1985; Moody et secrete al., 1985), large quantities of such bombesin peptides do not induce a phosphorylation response peptides (Moody et Erisman et al., 1981; al., 1982), and show identical with either PDGF or EGF suggests that there is not diminished proliferation in the presence of monoclonal antibodies a single common signal pathway which is activated by all these that bind specifically to the C terminus of bombesin and the mitogens. related peptides (Cuttitta et al., 1985). In order to understand words: Key bombesin/GRP/growth factors/protein-tyrosine kin- better the mechanism by which bombesin-related peptides may ase/tumor promoters be involved in tumor growth we have examined some early bombesin-mediated responses in Swiss 3T3 cells. Bombesin and the bombesin-related peptides are potent Introduction mitogens for Swiss 3T3 cells (Rozengurt and Sinnett-Smith, of The binding a number of such polypeptide growth 1983). This mitogenic factors, stimulation is exerted via their binding as platelet-derived factor to their cell- growth (PDGF), to a class of specific single about 100 000-300 000 high-affinity cell sur- surface in receptors causes an increase kinase protein-tyrosine face receptors per cell (Zachary and Rozengurt, 1985; Brown activity, which is to the bind- integral receptor. and Laurie, 1986). Binding is Following ligand accompanied by the breakdown ing, the receptor itself is in autophosphorylated and, of phosphoinositides (Brown et al., addition, 1984) and the subsequent a number of other substrate are proteins on activation of protein kinase C phosphorylated (Zachary et al., 1986) as measured in tyrosine and Hunter (reviewed Cooper and by increased phosphorylation Hunter, 1983; of a cellular protein of 80 000 of Treatment the cells with these Cooper, 1985). factors daltons, termed 80K (Rozengurt et growth al., 1983; Blackshear et al., in can also result the stimulation of serine and threonine 1985, 1986; Rodriguez-Pena and Rozengurt, 1986). We have of that phosphorylation target proteins. It has been proposed many confirmed these observations using a more direct assay for the of these latter events are due to the activation phosphorylation activation of protein kinase C, the phosphorylation of pp60csrc on serine 12 of a cellular kinase termed (Gould et al., We have also examined serine/threonine-specific 1985). the protein pro- tein kinase C. This would occur via the factor stimula- possibility that, as with other growth factors, the growth binding of © IRL Press 2889 Limited, Oxford, England et al. C.M.Isacke Table 1. Effect of ritogen treatment on incorporation of [32P]orthophosphate, inhibition of EGF binding, and [3H]thymidine incorporation in Swiss 3T3 cells Control Bombesin GRP PDGF EGF TPA PDBu Whole cell PAAa % Phosphotyrosine 0.031 0.039 0.044 0.334 0.038 0.044 0.039 % Phosphothreonine 8.75 8.52 8.08 9.26 8.71 9.26 9.82 % Phosphoserine 91.22 91.44 91.88 90.41 91.25 90.70 90.14 [3H]Thymidine incorporation x 4.1± 0.1 127 17 192 ± 30 611 37 190 i 37 202 i 14 120 14 (c.p.m. 103)b EGF binding (c.p.m.)c 1446 ± 91 459 ± 2 ND ND ND 99 10 95 ± 5 concentrations of mitogens: 1 nM PDGF, 10 nM bombesin, 10 nM GRP, 5 nM EGF, In all cases quiescent Swiss 3T3 cells were treated with the following 50 nM TPA, 300 nM PDBu. then treated for 15 min with aCells were labeled for 18 h in phosphate-free DMEM containing 5% (v/v) DMEM and 2 mCi/ml 32P-orthophosphate, and were mitogens prior to lysis. The levels of individual phosphoamino acids are expressed as a percentage of the total phosphoamino acid content. bDishes of quiescent cells were incubated for a further 24 h in DMEM alone. Mitogens were added with 1 insulin for a further 24 h and the cells were jig/ml labeled with for the final 6 h as described in Materials and methods. 10% FBS treatment resulted in a [3H]thymidine incorporation value of 493 [3H]thymidine + 47.5 x 103 c.p.m. Control values are those for cells cultured in the presence of insulin alone. Figures given are the mean values for three replicate samples. CCells were incubated for 18 h in phosphate-free DMEM containing 5% (v/v) DMEM and then treated for 60 min with mitogens and [125I]EGF, and then cell-associated [125I]EGF was measured as described in Materials and methods. Figures given are the mean value for three replicate samples. GRP to cells is by an increas- increase in the percentage of phosphate present in protein as bombesin and target accompanied kinase in addition to the elevation of phosphotyrosine. By contrast, treatment with saturating doses of ed protein-tyrosine activity resulted C activity. bombesin, GRP or either of the two tumor promoters protein kinase in only a marginal increase in the level of cellular phosphotyrosine was similar (Table I). The increment with the bombesin peptides Results to that with the tumor promoters and could be a consequence levels in vivo Changes in cellular phosphotyrosine of activation of protein kinase C (see below). In agreement with the phosphorylation events following bombesin treat- To examine published results, EGF treatment of Swiss 3T3 cells had little a clone of ment of intact cells, we used bombesin-responsive effect on the levels of cellular phosphotyrosine (Cooper et al., 100 000 bombesin Swiss 3T3 cells expressing > receptors per the fact that its receptor has ligand-stimulated 1982), despite Brown and cell and Rozengurt, 1985; Laurie, 1986). (Zachary protein-tyrosine kinase activity. for PDGF and These cells also have high-affinity receptors Phosphorylation of cellular protein-tyrosine kinase substrates and to these factor (EGF) respond mitogenically epidermal growth Brown et 1984; The effect of bombesin and GRP on the phosphorylation of factors (Collins and Rozengurt, 1984; al., Table Cells were cultured for 7 days cellular was examined in more detail by resolving Zachary et al., 1986; I). proteins and dishes were then treated in phosphoproteins from mitogen-treated cells in two-dimensional until they were quiescent parallel described below. This series of ex- We were interested in the phosphorylation of in a number of experiments gels. particularly on three occasions with similar three characterized p36, p42 and 80K. p36 was performed separate previously proteins, periments was initially identified as a major phosphoprotein in chicken cells results. or GRP to transformed with Rous sarcoma virus, which encodes the protein- we measured whether bombesin Initially binding tyrosine kinase, pp6ov-src (Radke and Martin, 1979). An increas- 3T3 cells was an elevation of cellular Swiss accompanied by pro- ed tyrosine phosphorylation of p36 in fibroblasts treated with levels similar to that described tein phosphotyrosine previously PDGF has been previously observed (Cooper et al., 1982; of these cells et For for PDGF treatment (Cooper al., 1982). Ralston and Bishop, 1985; Isacke et al., 1986). p42 is a low abun- of cells were either labeled for 18 h this dishes quiescent purpose, dance which is in mitogen-stimulated cells as a treated with for 15 min and protein present with [32P]orthophosphate, mitogens of related and pp42B) containing for whole-cell acid or pair phosphoproteins (pp42A then prepared phosphoamino analysis, as well as phosphoserine and in some cases for a further 24 h before addition of and phosphotyrosine, cultured mitogens (Cooper et al., 1984; Cooper and Hunter, 1 to measure stimulation of DNA phosphothreonine of insulin synthesis. Ag/ml 1985). The 80-kd phosphoproteins, which migrate at the acidic was a for these cells. In with PDGF potent mitogen agreement end of isoelectric focusing gels, have been identified as promi- results and 1982; previously published (Collins Rozengurt, et we found nent phosphorylation substrates in cells treated with both tumor and 1983; Corps al., 1985), Rozengurt Sinnett-Smith, with either GRP or promoters and growth factors (Rozengurt et al., 1983; Rodriguez- that treatment of Swiss 3T3 cells bombesin, in the of insulin Pena and Rozengurt, 1985; Bishop et al., 1985; Blackshear et the tumor TPA and PDBu, presence promoters, of al., 1985, 1986). It has been proposed that the phosphorylation resulted in a stimulation [3H]thymidine incorpora- significant serum-stimulated Table I of these proteins, referred to as 80K, is mediated by protein kinase tion of the maximal value; (25-40% for Swiss 3T3 C. and EGF was a moderately good mitogen legend). to Dishes of cells were labeled ovemight with [32P]orthophosphate cells incorporation approximately stimulating [3H]thymidine in with those assayed for whole-cell phosphoamino acid GRP and the tumor but parallel the same level as bombesin, promoters, levels lysed 15 min after the addition of less than serum or PDGF. (see Table I) and always little of the incor- mitogens. The phosphoproteins were separated by two-dimen- In resting cells very (-0.03%) phosphate sional with alkali was on residues. gel electrophoresis and the gels were then treated porated into cellular proteins present tyrosine to of the cells with PDGF sufficient to saturate the cell hydrolyse the majority of serine-bound phosphate groups. Treatment PDGF the for 15 to resulted in a 10-fold stimulated alkali-stable phosphorylation of a number surface receptors min prior lysis 2890 Bombesin- and GRP-induced phosphorylation events A FF_= B iWt: lb ,* 4p l-% 9 . aOK D.f _ r r l -042 -- w f so Control F EGF PDG ..iw a t w40i *9 9 I Bombesin GRP TPA Fig. 1. Two-dimensional gel electrophoresis of 32P-labeled proteins from mm mitogen-treated Swiss 3T3 cells. 35 dishes of quiescent Swiss 3T3 cells were labeled for 18 h with [32P]orthophosphate as described in Materials and methods and mitogens were added directly to the labeling medium for 15 min prior to lysis. (A) No addition, 1 (B) bombesin, 10 nM; (C) PDGF, nM; (D) TPA, 5 50 nM; (E) EGF, nM; (F) GRP, 10 nM. One-tenth of each sample was analysed on two-dimensional gels as described in Materials and methods. After fixation, gels were treated with alkali. Basic proteins are to the left of the gel; an approximate pH scale for the isoelectric focusing dimension is given in panel A. Arrowheads indicate the positions of pp42 (pointing left), pp36 (pointing right), and 80K (pointing upwards). Exposure times with an intensifying screen were 2 days. This experiment was performed in parallel with those described in Table I. of proteins including and 80K p36, p42 (Figure 1). Consistent seven discrete segments could be resolved in the 80K region. with previously published results, TPA treatment did not result To determine the number of phosphorylation sites in 80K and in any observable aLkali-stable p36 phosphorylation but did cause whether new sites were used upon mitogen stimulation, the whole dramatic increase in p42 phosphorylation and 80K phosphoryla- of group 32P-labeled 80K proteins was excised from untreated tion (Figure 1; see next section). PDBu treatment of cells induc- two-dimensional gels and subjected to phosphoamino acid analysis ed a series of phosphorylation events identical to that observed and two-dimensional tryptic peptide mapping (Figure 3). In quies- with TPA (data not shown). Two-dimensional resolution of the cent cells 80K contained phosphoserine and phosphothreonine phosphoproteins extracted from bombesin- or GRP-treated cells in a ratio of -4:1. The mitogen treatments tested resulted in a similar to gave pattern that from tumor promoter-treated cells, an increased serine phosphorylation of 80K with little change but the stimulation of 80K phosphorylation by bombesin or GRP in threonine phosphorylation and no detectable tyrosine phos- was less than that observed with the tumor promoters or PDGF, phorylation (Figure 3, insets). The presence of phosphothreonine while the stimulation of p42 phosphorylation was detectable but may explain the alkali-stability of 80K. Tryptic peptide maps weak (Figure 1). The increase in alkali-stable phosphorylation showed that 80K from untreated cells contained multiple of in tumor p42 promoter and bombesin- or GRP-treated cells phosphorylation sites which gave rise to 10 major tryptic in may explain part the small (but reproducible) increase in phosphopeptides. All the mitogen treatments stimulated the phos- cellular phosphotyrosine levels in these cells EGF (Table I). treat- phorylation of 80K at these same sites and at novel sites resulting ment resulted in an increase in p42 but not p36 phosphorylation. in the appearance of phosphopeptides 1, 3 and 5 (Figure 3, see Furthermore, no increase in 80K phosphorylation was observed schematic diagram in Figure 4). The mitogen-stimulated ap- suggesting that EGF treatment does not activate protein kinase pearance of the minor phosphopeptides, which migrated between C in these cells. peptides 7 and 11 and between peptides 8 and 10, was not reproducible (see Figure 4). Phosphorylation of 80K in mitogen-treated cells To examine the difference between the acidic and basic forms To obtain a quantitative estimate of the extent of 80K phos- of 80K, the phosphoproteins from untreated of gels mitogen- phorylation under various conditions, non-alkali treated were treated cells were excised in sections as indicated in gels Figure 2b, examined (Figure 2). It can be seen that 80K from mitogen-treated and subjected to peptide map analysis (Figure 4). Overall tryptic cells contained more acidic forms than that from untreated cells it was found that forms of 80K with similar from pI cells treated and that the most acidic of these tended to migrate more slowly in different ways had similar phosphopeptide patterns. For ex- than the most basic in the second dimension (Figure 2b). Up to ample, the most basic form of 80K from bombesin-treated cells 2891 et al. C.M.Isacke a b A B A 2,1 6 54 3 2 1 Bombesin Control C D 7C6,543 D 654 PDGF TPA -i Fig. 2. The 80K in two-dimensional from Swiss 3T3 cells. Two-dimensional of Swiss 3T3 cells were as region gels (a) gels [32P]orthophosphate-labeled described in 1 that the were dried before The mol. wt acidic of these 80K and Figure except gels fixing. nigher regions gels including surrounding proteins are shown. No 10 1 50 nM. time was 2 h with an screen. The same (A) addition, (B) bombesin, nM; (C) PDGF, nM; (D) TPA, Exposure intensifying (b) as shown in were and the 80K was the In the sections autoradiograms (a) enlarged region containing aligned vertically using addition, surrounding proteins. that were excised and subjected to in 4 are indicated numbers. tryptic peptide mapping Figure by a similar to the most basic form murine did not (B 1) had phosphopeptide pattern precipitated from cells [35S]methionine-labeled While of the with 80K from TPA-treated Swiss 3T3 in two- of 80K from untreated cells co-migrate cells (Control 1). many forms of and were in all the isoelectric dimensional gels phosphopeptides present (J.R.Woodgett J.Meisenhelder, unpublished 80K, most basic form of 80K some differences were noted. The results). (Con- 3 and whereas more trol 1 and B lacked 1) phosphopeptides 1, 5, Additional that bombesin activates kinase C evidence protein of these three acidic forms of the had a abundance protein greater forms of 80K a loss On toward the most acidic The activation of kinase C has been peptides. moving protein measured by a number of 8 and of 9 and 10 and 11 of indirect stimulation of 80K peptides (B 6) finally peptides assays including phosphorylation 6, was found. The data shown in 4 confirm the (TPA 7) and a of Figure (see above) decrease to its [1251]EGF binding receptor. in when the whole observation made Figure that Here we examined the in 3, namely, group [1251]EGF with binding capacity parallel of 80K are examined an increase in the amount of acidic proteins the and I and mitogenic phosphorylation asssays (Table Figure forms results in an increase in the amounts of 1). Dishes of cells were incubated for 60 with tryptic phospho- min and mitogens 3 and 5. Bombesin treatment resulted in a less com- peptides 1, [1251]EGF at 370C and the amount of radioactivity associated with conversion of basic to acidic forms of 80K than either PDGF the cells plete after this period was measured In (Table I). agreement or tumor treatments 2a and and has accor- with promoter (Figure previous reports all the mitogens tested caused a b) decrease less of 3 and 5 in EGF dingly peptides 1, binding (Brown et al., Sinnett-Smith (Figure 3). 1979; and It remained that 80K kinase C possible Rozengurt, 1985; Brown et Bombesin has represented protein al., 1984). been which is to have a mol. wt of 80 kd and be to be itself, reported reported as effective as PDGF in stimulating phospho- phosphorylated in intact cells et This inositide breakdown (Fry al., 1985). (Brown et in possibility al., 1984). However, our ex- was excluded that kinase C immuno- bombesin by demonstrating protein periments does not as an activator appear potent of 2892 Bombesin- and GRP-induced events phosphorylation *13 011 12 *X 4p8 40 4809 0.3 CONTROL BOMBESIN C D *13 p12 1-4 *7 4C . 6 _4 5 _.Je PDG TPA Fig. 3. Tryptic phosphopeptide maps and phosphoamino acid analysis of 80K from mitogen-treated Swiss 3T3 cells. The entire 80K area was excised from the two-dimensional in preparative gels shown Figure 2a and subjected to trypsin digestion and separation in two dimensions as described in Materials and methods. In each case the origin is marked (arrowhead). The peptide numbers correspond to those used in the schematic diagram in Figure 4. Electrophoresis on 100 cellulose was in the 1 thin-layer plates performed first dimension at pH 8.9 for 20 min at kV with the anode on the left. For each sample, 80K Am from 2-4 gels was pooled. Cerenkov c.p.m. in analysed each case were: (A) untreated cells, 1080 c.p.m.; (B) bombesin (10 nM), 420 c.p.m.; (C) PDGF (1 nM), 1330 c.p.m.; (D) TPA (50 nM), 1050 c.p.m. Inset: 1/8th of each 80K sample described above was subjected to phosphoamino acid analysis prior to trypsin digestion. The position of phosphoserine phosphothreonine (T) and (S), phosphotyrosine (Y) are indicated. Exposure time with an intensifying screen was 5 days. protein kinase C as either PDGF or TPA, based on the degree we examined the of in Swiss 3T3 cells phosphorylation pp6Oc-src of inhibition of EGF binding, as well as the lower level of p42 of in to treatments. The incubation response mitogen purified phosphorylation (Figure 1) and the fewer acidic forms of 80K kinase C with and in vitro or the protein pp6Oc-src [,y-32P]ATP generated (Figure 4; see Discussion). in both result in treatment of cells with tumor promoters vivo Finally, as neither the 80K phosphorylation nor EGF binding on a novel event et phosphorylation pp6Oc-src (Gould al, 1985; assays direct evidence for kinase C of 3T3 for 10 provide protein activation, Gentry et al, 1986). Treatment Swiss cells min 2893 C.M.Isacke et al. _13 *13 @13 13 _12 11 _ 12 _11 _10 1 11 11 12 8 a _ 9 4M8 _lm~ _7 ae 1pI* TPA 6 Contol i II _13 w 13 @13 _ 12 Z12 . 11 _011 40 11l _ _ 11 . 9 ; 8 8 9 12* 5_ B2 3 ' 9' I* _2 85 A B4 2 1 3331 B ; Fig. 4. Tryptic phosphopeptide maps of the acidic and basic forms of 80K from bombesin and 3T3 cells. Forms of 80K TPA, untreated Swiss with different isoelectric points were excised form preparative in 2b. gels as indicated Figure Trypsin digestion and mapping was as described in Materials and methods and in Figure 3. Maps from the most basic region of 80K from control cells (Control 1), five regions from bombesin-treated cells ranging from basic to acidic (B 1, B 2, B 3, B 4 and B 6) and three regions from TPA-treated cells (TPA 5, TPA 6 and TPA 7) are shown. For each sample 80K regions from six preparative two-dimensional gels were pooled. Cerenkov c.p.m. analysed and exposure time with an intensifying screen were: control 1, 400 c.p.m., 7 days; B 1, 210 7 B B B B c.p.m., days; 2, 620 c.p.m., 3 days; 3, 780 c.p.m., 3 days; 4, 1190 c.p.m., 3 days; 6, 430 c.p.m., 7 days; TPA 5, 1000 c.p.m., 3 TPA 1080 3 TPA 350 7 A of of is days; 6, c.p.m., days; 7, c.p.m., days. schematic diagram the tryptic phosphopeptides 80K shown. PDBu not PDGF or bombesin presence of ligand (Frackelton et al., 1984) or by Western blot- with either TPA, (data shown), stimulation of of on a ting of whole cell extracts following treatment with ligand (Zip- caused a large phosphorylation pp6oc-src residue to serine 12 in chicken pel et al., 1986). Although the bombesin receptor has not been equivalent pp6Oc-src (Figure 5), which gives rise to peptide 4. The other two characterized at the molecular level, Swiss 3T3 cells contain major pp60c-src observed in treated and untreated cells are enough bombesin receptors (- 100 000) for us to attempt to iden- phosphopeptides pep- tide which contains serine and which contains tify a protein-tyrosine kinase associated with its 1, 17, peptide 6, activity recep- Gould et et tor in this way. tyrosine 527 (Figure 5; al., 1985; Cooper al., 1986). The increased of serine 12 in To examine whether bombesin or GRP stimulate tyrosine phos- phosphorylation pp6Yc-src provides that bombesin treatment of Swiss 3T3 cells phorylation of any Swiss 3T3 cell membrane proteins, a crude further evidence of kinase C. EGF treat- membrane preparation was treated with bombesin, PDGF, or causes activation protein By contrast, in increase in EGF, incubated with and then ment did not result any pp6oc-src phosphorylation [,y-32P]ATP immunoprecipitated on 12 not that in these with anti-phosphotyrosine Ig. The EGF and PDGF receptors were serine (data shown) again demonstrating cells EGF does not activate kinase C. identified as 175 kd and 185 kd protein phosphoproteins respectively. In the described the PDGF used was a In bombesin-treated there was no detectable in experiments here, par- samples increase All these have been the phosphorylation of any membrane proteins (data not shown), tially pure preparation. experiments repeated at times with PDGF et and no different pure (Cooper al., 1982; C.M.I. phosphoprotein was specifically precipitated with K.L.G. In no case was dif- anti-phosphotyrosine Ig (Figure and unpublished 6a). We also analysed extracts observations). any ference in noted between the two PDGF of quiescent Swiss 3T3 cells treated with PDGF, EGF, response preparations. GRP or bombesin for 10-30 min by Western blotting with anti-phos- Detection with of growth factor receptors anti-phosphotyrosine photyrosine Ig. The 185 kd PDGF receptor was readily detected, antibodies in addition to of proteins 130, 70 and 33 kd (marked with ar- The failure to detect an increase in the level of phosphotyrosine rowheads in Figure 6), which were specific to PDGF-treated in total cellular or in known kinase proteins protein-tyrosine cells. The 175 kd EGF receptor was detectable in EGF-treated substrates does not the that the necessarily preclude possibility cells, but less apparent than the PDGF receptor. A protein of bombesin has intrinsic kinase receptor protein-tyrosine activity, 48 kd was in preferentially found EGF-treated cells (marked by since the bombesin have different to receptor might specificities arrowhead in In Figure 6). all the mitogen-treated samples pro- that of other known receptor protein-tyrosine kinases. Growth teins of 38 and 90 kd were observed, which were absent or less factor receptors, such as the PDGF which have receptor, intense in control samples. However, there were no striking bands associated protein-tyrosine kinase are on activity phosphorylated which were to extracts of unique bombesin- or GRP-treated cells tyrosine in the presence of their ligand. None of the techniques (Figure 6b). employed so far were designed specifically to detect autophos- of In phorylation the putative bombesin receptor. the absence Discussion of of specific anti-receptor antibodies, autophosphorylation growth factor receptors can be detected using anti-phospho- In examining phosphorylation events induced by bombesin and tyrosine antibodies either by immunoprecipitation of membrane GRP treatment of Swiss 3T3 cells, the most obvious changes are preparations phosphorylated in vitro by [-y-32P]ATP in the those which can be attributed to the activation of protein kinase 2894 Bombesin- and GRP-induced phosphorylation events CONTROL B BOMBESIN a- 1* 40 o. C PDGF D TPA A A~~~~~~~~~~~~~~~~~~~~~ A L- Fig. 5. Tryptic phosphopeptide maps of pp6O'-src from mitogen-treated Swiss 3T3 cells. pp6t-src was isolated from treated or untreated Swiss 3T3 cells by immunoprecipitation with MAb 327 (Lipsich et al., 1983), subjected to tryptic digestion and separated in two dimensions as described by Gould al. (1985). et In each case the origin is marked (arrowhead). Cells had been labeled with 2.5 mCi/ml [32P]orthophosphate for 18 h and treated for 10 140 nM 4-t- min with phorbol, (B) 10 nM bombesin, (C) 1 nM PDGF or (D) 75 nM TPA. Approximately 200 Cerenkov c.p.m. were analysed in each case. Exposure time an with screen was 5 intensifying days. C. Tumor which are to exert their confirmed that PDGF treatment, in addition to stimulating promoters, thought mitogenic effects to and kinase C activates in by binding activating protein (reviewed tyrosine phosphorylation, protein kinase C Swiss 3T3 in induce a number of cellular cells. By contrast we found no evidence that EGF induces stimula- Nishizuka, 1984; Ashendel, 1985), the indirect inhibition of EGF to its tion of kinase C. EGF events, including binding protein treatment failed to increase serine cell surface et Sinnett-Smith and 12 of receptor (Brown al., 1979; phosphorylation (data not shown) and does not pp6c-src and the increased of 80K stimulate 80K et Bishop Rozengurt, 1985) phosphorylation phosphorylation (Rozengurt al., 1983; et Blackshear et Rod- et Blackshear et (Rozengurt al., 1983; al., 1985, 1986; al., 1985; al., 1985; Figure 1). We routinely and Here we examined the ef- observed that neither bombesin nor GRP was as an efficient ac- riguez-Pena Rozengurt, 1986). fect of bombesin and GRP on these cellular events and found tivator of kinase C as the tumor GRP protein promoters, although that both criteria the bombesin activated kinase was reproducibly slightly more effective than bombesin. Tumor by peptides protein C 1 and thus the results of be more because are not as readily (Table I; Figures 2), confirming promoters may potent they Zachary et al. (1986). In addition, we employed a more direct metabolized as the DAG endogenously produced in response to for the activation of kinase the stimula- bombesin and GRP. assay protein C, namely Our of of ex- tion of serine 12 of et investigation the group 80 kd phosphoproteins phosphorylation pp6oc-src (Gould al., and showed that bombesin was able to induce this tends the work of others. Blackshear et al. (1985, 1986) 1985) phos- phorylation event (Figure 5). In the course of these studies we demonstrated that 80K from tumor PDGF or promoter, synthetic 2895 et al. C.M.Isacke a CEPB C B P E w - -_ *200 Il- iW, .1916 oil -'s. 116- i *97 - _ *68 681F 30* .30 with antibodies. For each a membrane from 6. Detection of factor anti-phosphotyrosine (a) sample crude preparation about 1 x l07 Fig. growth receptors 10 mM and incubated for 15 min at with to a concentration of 100 nM EGF Swiss 3T3 cells was resuspended in 20 Hepes (pH 7.4) 4°C mitogens give Al 1 or an volume of 1 mM acetic acid containing 1 mg/ml BSA as control (C) and then for (E), 100 nM PDGF (P), zM bombesin (B) equivalent (10 1l) as described in Materials and methods. The membranes were solubilized in RIPA buffer and immunoprecipitated 10 min at 30°C with 50 zICi [-y-32P]ATP antibodies as described in Materials and methods. One-half of each sample was resolved on a 10% SDS-polyacrylamide gel. with anti-phosphotyrosine at was 2 Swiss 3T3 cells in 35 mm dishes were treated with 10 nM bombesin (B), 1 nM Exposure time with intensifying screen -70°C days. (b) Quiescent 5 nM for or left untreated and then in SDS buffer. One-tenth of each sample was resolved on a 10% SDS- PDGF (P), EGF (E) 15 min (C) lysed sample immunoblotted as described in Materials and methods. time was 2 Mol. wt markers were (200 kd), 3- polyacrylamide gel and Exposure days. myosin bovine serum albumin ovalbumin kd) and carbonic anhydrase (30 kd). Arrowheads indicate galactosidase (116 kd), phosphorylase (97 kd), (68 kd), (43 proteins referred to in the text. on DAG-treated 3T3 cells was phosphorylated predominantly bands. to 13 were in pooled 80K Up phosphopeptides present serine residues, and resolved into several charge species. preparations. Because of the possibility of incomplete tryptic that like tumor Rozengurt and colleagues showed bombesin, pro- digestion, it is unclear exactly how many phosphorylation sites et moters, increased 80K phosphorylation (Zachary al., 1986) there are. If the charge isomers represent the addition of single and is to mediate this directly that protein kinase C very likely phosphate residues, then there can be as many as seven phos- 1986). By phosphoamino acid (Rodriguez-Pena and Rozengurt, phates per 80K molecule, implying there are at least seven distinct we found that 80K was analysis and tryptic peptide mapping phosphorylation sites. Some differences in tryptic phospho- on residues. Mitogen basally phosphorylated serine and threonine peptides content between the isoforms was evident in going from treatment caused additional phosphorylation on serine residues, basic to acidic, but there was no evidence that these phos- which in to more acidic forms. Seven resulted the conversion phorylations must occur in a strictly ordered sequence. Thus the isoelectric forms of resolved as discrete vertical 80K could be most basic form of 80K, which probably contains a single 2896 Bombesin- and GRP-induced phosphorylation events phosphate, can be phosphorylated at one of many sites. These their and induce distinct receptors yet phosphorylation responses results are consistent with there being a single 80K protein. that there are effector available implies multiple signal pathways The PDGF and EGF receptors have intrinsic ligand-stimulated for the kinases. receptor protein-tyrosine protein-tyrosine kinase activities. The fact that the response of To understand the role of bombesin and related peptides in Swiss 3T3 cells to bombesin has similarities to both the EGF an is whether the growth control, important question bombesin and PDGF responses, raises the question of whether the on Swiss 3T3 is the same bombesin cells as that on small receptor cell lung receptor might likewise have protein-tyrosine kinase activity. carcinoma A of We cells. the bombesin comparison and bombesin- failed to detect a marked increase in tyrosine related to these cell lines phosphorylation that the same peptide binding suggests of cellular proteins following bombesin treatment of is utilized in both cases quiescent and receptor (Zachary Rozengurt, 1985; cells, but the methods we used to detect such a stimulation have et or an of Moody al., 1985). [1251]GRP analogue bombesin con- limitations. For instance, only a minor increase in total phospho- an iodinatable taining tyrosine residue, [Tyr4-125I]bombesin, tyrosine levels is detected in cells following EGF treatment bind to a class of = unless single high affinity sites 0.5 In (KD nM). the cells have unusually high levels of EGF receptors such both cell this is with as bombesin-related types binding displaceable A431 cells (Hunter and Cooper, 1981). Thus even though which have Swiss peptides mitogenic activity, e.g., bombesin, (Tyr4) 3T3 cells possess about 85 000-100 000 EGF receptors bombesin and but not (Brown GRP, inactive such as vaso- by peptides et al., 1984; Zachary et al., 1986) and are mitogenically vasoactive intestinal respon- pressin, or substance P. an peptide Thus, sive to EGF (Table I), we did not observe a significant of the increase mechanism which the understanding by bombesin/GRP in phosphotyrosine levels following EGF treatment. on Two-dimen- receptor Swiss 3T3 cells transmits its mitogenic signal may sional gel analysis phosphoproteins of for well an phosphotyrosine- provide insight into the role of the bombesin-related pep- containing proteins also has shortcomings, which we tides in the autocrine have stimulation of small cell lung carcinomas. discussed elsewhere (Cooper and Hunter, 1983). For example, despite relatively the large increase in total phosphotyrosine in Materials and methods PDGF-treated cells, the only phosphotyrosine-containing proteins Materials detected by this technique are p42 and p36 (Cooper et al., 1982). PDGF 0.4% was a from ( pure) gift Elaine Raines (University of Washington, We were also unable to obtain direct evidence that the bom- Raines and Seattle, WA; EGF was obtained from Ross, 1982). S.Potter (The besin/GRP receptor has a ligand-stimulated protein-tyrosine Salk and Institute; Savage Cohen, 1972). 4-13-Phorbol, 4-3-phorbol-12,13-di- kinase activity, by using anti-phosphotyrosine and antibodies butyrate 12-O-tetradecanoyl-phorbol-13-acetate either (PDBu) (TPA) were obtain- ed from Chemicals. Sigma Bombesin and GRP were a for immunoprecipitation gift from Jean Rivier of in vitro phosphorylated (The membrane Salk Institute; Rivier and Brown, 1978). Peptides were dissolved in water or preparations or for Western blotting of whole-cell extracts, techni- Dulbecco-Vogt modified Eagle's medium (DMEM) and stored 1 as mM stock ques which allowed detection of the PDGF receptor. This con- solutions at -20°C. Concentrations of mitogens used in each assay are given trasts with a recent study by Comoglio and co-workers in the (Cirillo figure legends. et al., 1986), where a 105 kd protein was observed by Western Cells blotting extracts bombesin-treated of Swiss 3T3 cells with bombesin-responsive Swiss 3T3 cell clone was obtained from Enrique Rozengurt anti-phosphotyrosine Ig. These workers were also able to im- (ICRF, London), and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). munoprecipitate Unless otherwise stated confluent, quiescent cells a protein of this size, whose were prepared phosphorylation for x assays by seeding 2.5 101 cells into a 35 mm dish in DMEM on tyrosine supplemented in vitro was stimulated by bombesin. Their results with 10% FBS and culturing for 6-7 days. For metabolic labeling, the medium suggest that the bombesin receptor is a protein of 105 kd with was then aspirated and the cells cultured for a further 18 h in 1 ml of phosphate- ligand-stimulated protein-tyrosine kinase activity. Our failure to free DMEM containing 5% (v/v) DMEM and 2 mCi of [32P]orthophosphate detect such a protein may be attributable to the (ICN). Mitogens were added for 15 min prior to anti-phospho- lysis. A crude membrane preparation was made from tyrosine antibody preparation Swiss 3T3 cells as follows: we used, since there is documented cells - ( 80% confluent) were scraped from mm 150 dishes at 4°C in 2 ml of variability in the affinity of anti-phosphotyrosine antibodies 5 mM Hepes (pH 7.4), 2 mM MgCl2, 5 mM 2-mercaptoethanol, 0.1% aprotinin, towards individual phosphotyrosine-containing proteins. 0.1 mM 4 PMSF, 4 SBTI, leupeptin and homogenized with 25 jug/ml ;sg/ml If the bombesin receptor has protein-tyrosine kinase strokes in activity, a Dounce homogenizer using a tight fitting pestle. The homogenate was centrifuged at 200 for 5 mins to remove remaining whole cells, our analysis of tyrosine phosphorylation events nuclei in the intact g and cell cell debris, and then at 20 000 g for 60 min. The supematant was discarded and suggests that the bombesin receptor either has a lower specific the membrane pellet stored in liquid N2. activity or else has a much more restricted substrate specificity Assays than the PDGF receptor protein-tyrosine kinase. In this respect Whole cell phosphoamino acid analysis. This was performed according to Sef- the properties of the bombesin receptor are more akin to those ton et al. (1980). of the EGF receptor, since treatment by neither factor results Two-dimensional gel electrophoresis. This was performed using the method of in elevation an in whole-cell phosphotyrosine levels. However, Garrels (1979) modified by Cooper and Hunter (1981) except that isoelectric focus- a major difference in the mechanism of action of the was bombesin ing with pH 3.5-10 ampholytes (LKB). The gels were fixed, dried and rehydrated 1 and EGF receptors is observed; while PDGF and in M KOH, incubated at 550C for 2 h, neutralized, bombesin and dried again are (Cooper and Hunter, 1981). Preparative gels for 80K were rinsed for 30 min potent activators of PI turnover in Swiss 3T3 cells (Habenicht in water with mixed-bed ion resin exchange (Amberlite) and dried without fixing. et al., 1981; Brown et al., 1984), EGF treatment of most cells, Inhibition ofEGF binding. This was to assayed according Brown et al. (1984). including Swiss 3T3 cells, does not result in the activation of Essentially dishes of cells were cultured overnight in labeling medium without protein kinase C (Bishop et al., 1985; Blackshear et al., 1985; [32P]orthophosphate and then incubated at 370C for 60 min in 1 ml DMEM con- Habenicht et al., 1981; Coughlin et al., 1985). Clearly one needs taining mitogens and 27 000 c.p.m. [125I]EGF (NEN; 175 After washing jiCi/4g). in ice-cold to identify the substrate for the PDGF receptor DMEM, the cells were extracted with 0.5 M NaOH and protein-tyrosine counted in a y-counter. kinase which when phosphorylated is responsible for the increase Measurement of [3H]thymidine incorporation. Dishes of cells were in DAG level, and determine whether this cultured for is also phosphorylated a further 24 h with 2 ml of DMEM. Mitogens together with 1 in insulin were bombesin-treated cells, or whether itg/ml bombesin activates PT turn- added for another 24 h, and the cells were labeled with 2 [methyl- isCi/ml over in another manner. The fact that PDGF, EGF and probably 3H]thymidine (6.7 Ci/mmol, NEN) for the final 6 h. Cells were then fixed with bombesin all stimulate tyrosine phosphorylation upon binding to methanol:acetic acid (3:1, v/v), washed with 10% extensively trichloroacetic acid 2897 C.M.Isacke et al. (TCA) at 4°C, rinsed with PBS and solubilized in 1 ml 0.2 M NaOH, 0.5 ml Cooper,J.A. and Hunter,T. (1981) Mol. Cell. Biol., 1, 165-178. was counted in 10 ml of aqueous scintillation fluid. Cooper,J.A. and Hunter,T. (1983) Curr. Topics Microbiol. Immunol., 107, 125- 161. Peptide mapping and phosphoamino acid analysis. 80K phosphoproteins were Mol. Cell. Cooper,J.A. and Hunter,T. (1985) Biol., 5, 3304-3309. excised from the preparative gels and then protein was subjected to phosphoamino Cooper,J.A, Bowen-Pope,D.F., Raines,E., and Hunter,T. acid analysis or tryptic peptide mapping as described by Hunter and Sefton (1980). Ross,R. (1982) Cell, 263-273. 31, The 32P-labeling of pp6(j-src in vivo and its tryptic peptide mapping have been Cooper,J.A., Sefton,B.M. and Hunter,T. (1984) previously described Mol. Cell. Biol., 4, 30-37. (Gould et al., 1985). In this experiment mitogen treatments Cooper,J.A., Gould,K.L., Cartwright,C.A. and Hunter,T. (1986) were for 10 min Science, 231, prior to lysis. 1431-1433. Western blotting. Dishes of cells were cultured for a further 24 h in DMEM alone Corps,A.N., Rees,L.H. and Brown,K.D. (1985) Biochem. J., 231, 781-784. and then treated with mitogens for 10 to 30 min before rinsing once with cold Coughlin,S.R., Lee,W.M.F., Williams,P.W., Giels,G.M. and Williams,L.T. PBS and lysing the cells in 0.3 ml boiling Laemmli sample buffer. The samples (1985) Cell, 43, 243-251. were boiled for 5 min, sheared by repeated passage through a 27 gauge needle, Cuttitta,F., Carney,D.N., Mulshine,J., Moody,T.W., Fedorko,J., Fischler,A. reboiled for 20 min and equal volumes were resolved on 10% SDS-polyacryla- and Minna,J.D. (1985) Nature, 316, 823-826. mide gels. Proteins were transferred to 0.45 nitrocellulose (Schleicher itm and Erisman,M.D., Linnoila,R.I., Hernandez,O., DiAugustine,R.P. and Lazarus,L.H. Schuell) in a BioRad apparatus using a Tris-glycine buffer containing 20% (1982) Proc. Natl. Acad. Sci. USA, 79, 2379-2383. methanol at a current of 40 V for 90 min. The blot was then blocked overnight Frackelton,A.R.,Jr, Tremble,P. and Williams,L.T. (1984) J. Biol. Chem., 259, in rinse buffer (10 mM pH 7.4, 0.15 M NaCI) containing 5% BSA Tris-HCI, 7907-7914. and 1% ovalbumin and probed for 2 h in same buffer containing the 4 Fry,M.J., Gebhardt,A., Parker,P. and Foulkes,J.G. Agg/ml (1985) EMBO J., 4, affinity-purified anti-phosphotyrosine Ig generated by the immunization of rab- 3173-3178. bits with poly[phosphotyrosine.Ala.Gly] covalently linked to keyhold limpet hemo- Garrels,J.I. (1979) J. Biol. Chem., 254, 7961-7977. cyanin (a kind gift of Mark Kamps, Salk Institute). The blot was then washed Gentry,L.E., Chaffin,K.E., Shoyab,M. and Purchio,A.F. (1986) Mol. Cell. Biol., twice in rinse buffer, once in rinse buffer containing 0.05% NP-40, and then 6, 735-738. twice more in rinse buffer for 10 min each. The blot was incubated with [1251]- Gilmore,T. and Martin,G.S. (1983) Nature, 306, 487-490. protein A for 1 h to decorate bound antibody, washed as above, dried and ex- Gould,K.L., Woodgett,J.R., Cooper,J.A., Buss,J.E., Shalloway,D. and Hunter,T. posed to film. (1985) Cell, 42, 849-857. Immunoprecipitation of phosphorylated membrane proteins. Crude membrane Habenicht,A.J., Glomset,J.A., King,W.C., Nist,C., Mitchell,C. and Ross,R. - 1 preparations (see above) from x 107 cells were resuspended in 20 of 1I (1981) J. Biol. Chem., 256, 12329-12335. 10 mM Hepes (pH 7.4), incubated for 15 min at 4°C with mitogens and then Hunter,T. and Cooper,J.A. (1981) Cell, 24, 741-752. phosphorylated in vitro for 10 min at 30°C with 50 /Ci [-y-32P]ATP Hunter,T. and Cooper,J.A. (1985) Ann. Rev. Biochem., 54, 897-930. (3000 Ci/mmol, Amersham) in 25 of 20 mM Pipes (pH 7.4), 10 mM MnCl2, Hunter,T. and Sefton,B.M. (1980) Proc. Natl. Acad. Sci. USA, 77, 1311-1315. A1 4 unlabeled ATP (final concentration). Membranes were then solubilized in Isacke,C.M., Trowbridge,I.S. and Hunter,T. (1986) Mol. Cell. ytM Biol., 6, 0.5 ml RIPA buffer (Sefton et al., 1978) containing 50 mM Tris, pH 7.4 in- 2745-2751. stead of phosphate buffer and supplemented with mM EDTA; 100 5 u1M Na3VO4; Lipsich,L.A., Lewis,A.J. and Brugge,J.S. (1983) J. Virol., 48, 352-360. 50 mM NaF. The lysate was then clarified by centrifugation at 20 000 g for 60 min McDonald,T.J., Jornvall,H., Nilsson,G., Vagne,M., Ghatei,M., Bloom,S.R. and and immunoprecipitated according to Sefton et al. (1978) using anti-phospho- Mutt,V. (1979) Biochem. Biophys. Res. Commun., 90, 227-233. tyrosine Ig affinity-purified from a rabbit antiserum raised against phosphotyrosine Moody,T.W., Pert,C.B., Gazdar,A.F., Carney,D.N. and Minna,J.D. (1981) coupled to BSA (obtained from Mark Kamps, The Salk Institute). Science, 214, 1246-1248. Moody,T.W., Carney,D.N., Cuttitta,F., Quattrocchi,K. and Minna,J.D. (1985) Autoradiography. This was performed using Kodak XAR film, which where in- Life Sci., 37, 105-113. dicated was pre-sensitized and exposed at -70°C with an intensifying screen. Nishizuka,Y. (1984) Nature, 308, 693-698. Radke,K. and Martin,G.S. (1979) Proc. Natl. Acad. Sci. USA, 76, 5212-5216. Acknowledgements Raines,E. and Ross,R. (1982) J. Biol. Chem., 257, 5154-5160. We are extremely grateful to Enrique Rozengurt for providing us with Ralston,R. and Bishop,J.M. (1985) Proc. Natl. Acad. Sci. the USA, 82, 7845-7849. bombesin-responsive clone of Swiss 3T3 cells and also communicating Rivier,C., Rivier,J.E. and Vale,W. for results (1978) Endocrinol., 102, 519-524. prior to publication, and to Paolo Comoglio for informing us of experiments Rivier,J.E. and Brown,M.R. (1978) Biochemistry, con- 17, 1766-1771. cerning the nature Rodriguez-Pena,A. and of the bombesin receptor prior to publication. We thank Jean Rozengurt,E. (1985) EMBO J., 4, 71-76. Rivier for supplying bombesin and gastrin-releasing peptide and Mark Rodriguez-Pena,A. Kamps and Rozengurt,E. (1986) EMBO J., 5, 77-83. for the affinity-purified anti-phosphotyrosine antibodies. This work was supported Rozengurt,E. and Sinnett-Smith,J. (1983) Proc. Natl. Acad. Sci. USA, 80, by Public Health Service Grant Numbers 2936-2940. CA 17096, CA 28458 and CA 39780 Rozengurt,E., from the National Cancer Institute. C.M.I. was a recipient of a fellowship from Rodriguez-Pena,M. and Smith,K.A. (1983) Proc. Natl. Acad. Sci. the Damon Runyon-Walter Winchell Cancer Fund (DRG-775). K.D.B. was USA, 80, 7244-7248. a recipient of a grant from the Nuffield Foundation and is grateful to Bob Holley Savage,C.R.,Jr and Cohen,S. (1972) J. Biol. Chem., 247, 7609-7611. at the Salk Institute for providing support and laboratory space. K.L.G. and S.J.G. Sefton,B.M., Beemon,K. and Hunter,T. (1978) J. Virol., 28, 957-971. were supported by National Institutes of Health predoctoral training grants to Sefton,B.M., Hunter,T., Beemon,K. and Eckhart,W. (1980) Cell, 20, 807-816. the University of California at San Diego. Sinnett-Smith,J.W. and Rozengurt,E. (1985) J. Cell. Physiol., 124, 81-86. Swope,S.L. and Schonbrunn,A. (1984) Proc. Natl. Acad. Sci. USA, 81, 1822-1826. References Weber,S, Zuckerman,J.E., Bostwick,D.G., Bensch,K.G., Sikic,B.I. and Raffin, Anastasi,A., Erpsamer,V. and Bucci,M. (1971) Experientia, 27, 166-167. (1985) J. Clin. T.A. Invest., 75, 306-309. Ashendel,C.L. (1985) Biochem. Biophys. Acta, 822, 219-242. Westendorf,J.M. and Schonbrunn,A. (1982) Endocrinology, 110, 352-358. Blackshear,P.J., Witters,L.A., Girard,P.R., Kuo,J.F. and Quamo,S.N. (1985) Westendorf,J.M. and Schonbrunn,A. (1983) J. Biol. Chem., 258, 7527-7535. J. Biol. Chem., 260, 13304-13315. Willey,J.C., Lechner,J.F. and Harris,C.C. (1984) Exp. Cell Res., 153, 245-248. Blackshear,P.J., Wen,L., Glynn,B.P. and Witters,L. (1986) J. Biol. Chem., 261, Zachary,I. and Rozengurt,E. (1985) Proc. Natl. Acad. Sci. USA, 82,7616-7620. 1459-1469. Zachary,I., Sinnett-Smith,J.W. and Rozengurt,E. (1986) J. Cell Biol., 102, Bishop,R., Martinez,R., Weber,M.J., Blackshear,P.J., Beatty,S., Lim,R. and 2211 -2221. Herschman,H.R. (1985) Mol. Cell. 5, 2231-2237. Biol., Zippel,R., Sturani,E., Toschi,L., Naldin,L., Alberghina,L. and Comoglio,P.M. Brown,K.D. Laurie,M.S. and (1986) J. Physiol., 71, p. 210. (1986) Biochem. Biophys. Acta, 881, 56-61. Brown,K.D., Dicker,P. and Rozengurt,E. (1979) Biochem. Biophys. Res. Commun., 86, 1037-1043. Received on 2 April 1986; 1 revised on September 1986 Brown,K.D., Blay,J., Irvine,R.F., Heslop, J.P. and Berridge,M.J. (1984) Biochem. Biophys. Res. Commun., 123, 377-384. Brown,M., Marki,W. and Rivier,J. (1980) Life Sci., 27, 125-128. Cirillo,D.M., Gaudino,G., Naldini,L. and Comoglio,P.M. (1986) Mol. Cell. Biol., in press. Collins,M.K.L. and Rozengurt,E. (1982) J. Cell. Physiol., 112, 42-50. Collins,M.K.L. and Rozengurt,E. (1984) J. Cell. Physiol., 118, 133-142. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin‐related peptide, gastrin‐releasing peptide.

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Copyright © European Molecular Biology Organization 1986
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10.1002/j.1460-2075.1986.tb04584.x
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Abstract

The EMBO Journal vol.5 no. 11 -2898, pp.2889 1986 Early events phosphorylation the treatment of Swiss following 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide Clare M.Isacke1, Jill tion Meisenhelder', Kenneth of D.Brown2, phosphatidylinositol (PI) breakdown, which a generates Kathleen L.Gould13, Stephen J.Gould3 and Tony transient Hunter' intermediate, that binds to and diacylglycerol (DAG), activates kinase C in protein (reviewed Nishizuka, 1984; 'Molecular Biology and Virology Laboratory, The Salk Institute, PO Box Protein kinase C also acts as a cellular 85800, San Diego, Ashendel, 1985). major CA 92138, USA, 2AFRC Institute of Animal Physiology, for tumor such as Babraham, Cambridge CB2 4AT, UK and receptor promoters 3Department of 12-O-tetradecanoyl-phor- Biology, University of California at San Diego, La Jolla, CA 92093, USA bol-13-acetate and (TPA) phorbol dibutyrate which can (PDBu), substitute for DAG in the Communicated activation of the by R.Dulbecco enzyme. Thus, activ- ation of kinase C is protein to an essential role in likely play the Bombesin and the related mammalian peptides, such as stimulation elicited mitogenic tumor by promoters (reviewed in gastrin-releasing peptide (GRP), are potent mitogens for some Nishizuka, 1984; Ashendel, 1985). fibroblast cell lines. Here we have examined the bombesin- Bombesin is a tetradecapeptide isolated from the skin of Bom- and GRP-mediated changes in the phosphorylation of pro- bina bombina et Several (Anastasi al., 1971). peptides structurally teins in Swiss 3T3 cells and compared these to the events related to bombesin have been isolated from mammalian tissue observed after platelet-derived growth factor (PDG1F), epider- and are they characterized a N by having pyroglutamyl terminus mal growth factor (EGF) and tumor promoter treatment. In and a highly homologous C-terminal octapeptide. The best agreement with previous reports, bombesin, GRP and PDGF, characterized is gastrin-releasing peptide (GRP; McDonald et but not al., EGF, increased the activity of protein kinase C. This which 1979), with bombesin for to competes binding high-affinity was assayed by an inhibition of binding, stimula- [1251]EGF cell surface receptors on cells target (Westendorf and Schon- tion in phosphorylation of pp6oc-ws on serine 12 and stimula- brunn, and and elicits 1983; Zachary Rozengurt, 1985) identical tion in phosphorylation of a group of 80 kd proteins. The when responses injected into experimental animals (Brown et al., different phosphorylated forms of the 80 kd proteins were The 1980). bombesin-related peptides have a wide range of examined by tryptic peptide mapping and shown to contain physiological effects including the stimulation of hormone release multiple phosphorylation sites. An investigation of the tyrosine in vivo and in vitro (e.g. Rivier et al., 1978; Westendorf and phosphorylation events following mitogen treatment reveal- Schonbrunn, and 1982; Swope Schonbrunn, 1984) and stimula- ed a significant difference between PDGF and the bombesin tion of cell division in cultured fibroblasts (Rozengurt and Sinnett- peptides. PDGF treatment caused a marked increase in total Smith, 1983) and human bronchial epithelial cells (Willey et al., cellular phosphoyrosine levels, and trosine phosphorylation 1984). both of known substrates and its own receptor. In contrast, it Recently has been that suggested bombesin-related peptides bombesin and GRP treatments resulted in only a weak or are involved in the autocrine stimulation of human small-cell lung undetectable increase in tyrosine phosphorylation of total carcinomas as these cells respond mitogenically to and express cellular protein or known substrates. In this respect bombesin high-affinity receptors for bombesin-related peptides (Weber et and GRP were more similar to EGF. The fact that the al., 1985; Moody et secrete al., 1985), large quantities of such bombesin peptides do not induce a phosphorylation response peptides (Moody et Erisman et al., 1981; al., 1982), and show identical with either PDGF or EGF suggests that there is not diminished proliferation in the presence of monoclonal antibodies a single common signal pathway which is activated by all these that bind specifically to the C terminus of bombesin and the mitogens. related peptides (Cuttitta et al., 1985). In order to understand words: Key bombesin/GRP/growth factors/protein-tyrosine kin- better the mechanism by which bombesin-related peptides may ase/tumor promoters be involved in tumor growth we have examined some early bombesin-mediated responses in Swiss 3T3 cells. Bombesin and the bombesin-related peptides are potent Introduction mitogens for Swiss 3T3 cells (Rozengurt and Sinnett-Smith, of The binding a number of such polypeptide growth 1983). This mitogenic factors, stimulation is exerted via their binding as platelet-derived factor to their cell- growth (PDGF), to a class of specific single about 100 000-300 000 high-affinity cell sur- surface in receptors causes an increase kinase protein-tyrosine face receptors per cell (Zachary and Rozengurt, 1985; Brown activity, which is to the bind- integral receptor. and Laurie, 1986). Binding is Following ligand accompanied by the breakdown ing, the receptor itself is in autophosphorylated and, of phosphoinositides (Brown et al., addition, 1984) and the subsequent a number of other substrate are proteins on activation of protein kinase C phosphorylated (Zachary et al., 1986) as measured in tyrosine and Hunter (reviewed Cooper and by increased phosphorylation Hunter, 1983; of a cellular protein of 80 000 of Treatment the cells with these Cooper, 1985). factors daltons, termed 80K (Rozengurt et growth al., 1983; Blackshear et al., in can also result the stimulation of serine and threonine 1985, 1986; Rodriguez-Pena and Rozengurt, 1986). We have of that phosphorylation target proteins. It has been proposed many confirmed these observations using a more direct assay for the of these latter events are due to the activation phosphorylation activation of protein kinase C, the phosphorylation of pp60csrc on serine 12 of a cellular kinase termed (Gould et al., We have also examined serine/threonine-specific 1985). the protein pro- tein kinase C. This would occur via the factor stimula- possibility that, as with other growth factors, the growth binding of © IRL Press 2889 Limited, Oxford, England et al. C.M.Isacke Table 1. Effect of ritogen treatment on incorporation of [32P]orthophosphate, inhibition of EGF binding, and [3H]thymidine incorporation in Swiss 3T3 cells Control Bombesin GRP PDGF EGF TPA PDBu Whole cell PAAa % Phosphotyrosine 0.031 0.039 0.044 0.334 0.038 0.044 0.039 % Phosphothreonine 8.75 8.52 8.08 9.26 8.71 9.26 9.82 % Phosphoserine 91.22 91.44 91.88 90.41 91.25 90.70 90.14 [3H]Thymidine incorporation x 4.1± 0.1 127 17 192 ± 30 611 37 190 i 37 202 i 14 120 14 (c.p.m. 103)b EGF binding (c.p.m.)c 1446 ± 91 459 ± 2 ND ND ND 99 10 95 ± 5 concentrations of mitogens: 1 nM PDGF, 10 nM bombesin, 10 nM GRP, 5 nM EGF, In all cases quiescent Swiss 3T3 cells were treated with the following 50 nM TPA, 300 nM PDBu. then treated for 15 min with aCells were labeled for 18 h in phosphate-free DMEM containing 5% (v/v) DMEM and 2 mCi/ml 32P-orthophosphate, and were mitogens prior to lysis. The levels of individual phosphoamino acids are expressed as a percentage of the total phosphoamino acid content. bDishes of quiescent cells were incubated for a further 24 h in DMEM alone. Mitogens were added with 1 insulin for a further 24 h and the cells were jig/ml labeled with for the final 6 h as described in Materials and methods. 10% FBS treatment resulted in a [3H]thymidine incorporation value of 493 [3H]thymidine + 47.5 x 103 c.p.m. Control values are those for cells cultured in the presence of insulin alone. Figures given are the mean values for three replicate samples. CCells were incubated for 18 h in phosphate-free DMEM containing 5% (v/v) DMEM and then treated for 60 min with mitogens and [125I]EGF, and then cell-associated [125I]EGF was measured as described in Materials and methods. Figures given are the mean value for three replicate samples. GRP to cells is by an increas- increase in the percentage of phosphate present in protein as bombesin and target accompanied kinase in addition to the elevation of phosphotyrosine. By contrast, treatment with saturating doses of ed protein-tyrosine activity resulted C activity. bombesin, GRP or either of the two tumor promoters protein kinase in only a marginal increase in the level of cellular phosphotyrosine was similar (Table I). The increment with the bombesin peptides Results to that with the tumor promoters and could be a consequence levels in vivo Changes in cellular phosphotyrosine of activation of protein kinase C (see below). In agreement with the phosphorylation events following bombesin treat- To examine published results, EGF treatment of Swiss 3T3 cells had little a clone of ment of intact cells, we used bombesin-responsive effect on the levels of cellular phosphotyrosine (Cooper et al., 100 000 bombesin Swiss 3T3 cells expressing > receptors per the fact that its receptor has ligand-stimulated 1982), despite Brown and cell and Rozengurt, 1985; Laurie, 1986). (Zachary protein-tyrosine kinase activity. for PDGF and These cells also have high-affinity receptors Phosphorylation of cellular protein-tyrosine kinase substrates and to these factor (EGF) respond mitogenically epidermal growth Brown et 1984; The effect of bombesin and GRP on the phosphorylation of factors (Collins and Rozengurt, 1984; al., Table Cells were cultured for 7 days cellular was examined in more detail by resolving Zachary et al., 1986; I). proteins and dishes were then treated in phosphoproteins from mitogen-treated cells in two-dimensional until they were quiescent parallel described below. This series of ex- We were interested in the phosphorylation of in a number of experiments gels. particularly on three occasions with similar three characterized p36, p42 and 80K. p36 was performed separate previously proteins, periments was initially identified as a major phosphoprotein in chicken cells results. or GRP to transformed with Rous sarcoma virus, which encodes the protein- we measured whether bombesin Initially binding tyrosine kinase, pp6ov-src (Radke and Martin, 1979). An increas- 3T3 cells was an elevation of cellular Swiss accompanied by pro- ed tyrosine phosphorylation of p36 in fibroblasts treated with levels similar to that described tein phosphotyrosine previously PDGF has been previously observed (Cooper et al., 1982; of these cells et For for PDGF treatment (Cooper al., 1982). Ralston and Bishop, 1985; Isacke et al., 1986). p42 is a low abun- of cells were either labeled for 18 h this dishes quiescent purpose, dance which is in mitogen-stimulated cells as a treated with for 15 min and protein present with [32P]orthophosphate, mitogens of related and pp42B) containing for whole-cell acid or pair phosphoproteins (pp42A then prepared phosphoamino analysis, as well as phosphoserine and in some cases for a further 24 h before addition of and phosphotyrosine, cultured mitogens (Cooper et al., 1984; Cooper and Hunter, 1 to measure stimulation of DNA phosphothreonine of insulin synthesis. Ag/ml 1985). The 80-kd phosphoproteins, which migrate at the acidic was a for these cells. In with PDGF potent mitogen agreement end of isoelectric focusing gels, have been identified as promi- results and 1982; previously published (Collins Rozengurt, et we found nent phosphorylation substrates in cells treated with both tumor and 1983; Corps al., 1985), Rozengurt Sinnett-Smith, with either GRP or promoters and growth factors (Rozengurt et al., 1983; Rodriguez- that treatment of Swiss 3T3 cells bombesin, in the of insulin Pena and Rozengurt, 1985; Bishop et al., 1985; Blackshear et the tumor TPA and PDBu, presence promoters, of al., 1985, 1986). It has been proposed that the phosphorylation resulted in a stimulation [3H]thymidine incorpora- significant serum-stimulated Table I of these proteins, referred to as 80K, is mediated by protein kinase tion of the maximal value; (25-40% for Swiss 3T3 C. and EGF was a moderately good mitogen legend). to Dishes of cells were labeled ovemight with [32P]orthophosphate cells incorporation approximately stimulating [3H]thymidine in with those assayed for whole-cell phosphoamino acid GRP and the tumor but parallel the same level as bombesin, promoters, levels lysed 15 min after the addition of less than serum or PDGF. (see Table I) and always little of the incor- mitogens. The phosphoproteins were separated by two-dimen- In resting cells very (-0.03%) phosphate sional with alkali was on residues. gel electrophoresis and the gels were then treated porated into cellular proteins present tyrosine to of the cells with PDGF sufficient to saturate the cell hydrolyse the majority of serine-bound phosphate groups. Treatment PDGF the for 15 to resulted in a 10-fold stimulated alkali-stable phosphorylation of a number surface receptors min prior lysis 2890 Bombesin- and GRP-induced phosphorylation events A FF_= B iWt: lb ,* 4p l-% 9 . aOK D.f _ r r l -042 -- w f so Control F EGF PDG ..iw a t w40i *9 9 I Bombesin GRP TPA Fig. 1. Two-dimensional gel electrophoresis of 32P-labeled proteins from mm mitogen-treated Swiss 3T3 cells. 35 dishes of quiescent Swiss 3T3 cells were labeled for 18 h with [32P]orthophosphate as described in Materials and methods and mitogens were added directly to the labeling medium for 15 min prior to lysis. (A) No addition, 1 (B) bombesin, 10 nM; (C) PDGF, nM; (D) TPA, 5 50 nM; (E) EGF, nM; (F) GRP, 10 nM. One-tenth of each sample was analysed on two-dimensional gels as described in Materials and methods. After fixation, gels were treated with alkali. Basic proteins are to the left of the gel; an approximate pH scale for the isoelectric focusing dimension is given in panel A. Arrowheads indicate the positions of pp42 (pointing left), pp36 (pointing right), and 80K (pointing upwards). Exposure times with an intensifying screen were 2 days. This experiment was performed in parallel with those described in Table I. of proteins including and 80K p36, p42 (Figure 1). Consistent seven discrete segments could be resolved in the 80K region. with previously published results, TPA treatment did not result To determine the number of phosphorylation sites in 80K and in any observable aLkali-stable p36 phosphorylation but did cause whether new sites were used upon mitogen stimulation, the whole dramatic increase in p42 phosphorylation and 80K phosphoryla- of group 32P-labeled 80K proteins was excised from untreated tion (Figure 1; see next section). PDBu treatment of cells induc- two-dimensional gels and subjected to phosphoamino acid analysis ed a series of phosphorylation events identical to that observed and two-dimensional tryptic peptide mapping (Figure 3). In quies- with TPA (data not shown). Two-dimensional resolution of the cent cells 80K contained phosphoserine and phosphothreonine phosphoproteins extracted from bombesin- or GRP-treated cells in a ratio of -4:1. The mitogen treatments tested resulted in a similar to gave pattern that from tumor promoter-treated cells, an increased serine phosphorylation of 80K with little change but the stimulation of 80K phosphorylation by bombesin or GRP in threonine phosphorylation and no detectable tyrosine phos- was less than that observed with the tumor promoters or PDGF, phorylation (Figure 3, insets). The presence of phosphothreonine while the stimulation of p42 phosphorylation was detectable but may explain the alkali-stability of 80K. Tryptic peptide maps weak (Figure 1). The increase in alkali-stable phosphorylation showed that 80K from untreated cells contained multiple of in tumor p42 promoter and bombesin- or GRP-treated cells phosphorylation sites which gave rise to 10 major tryptic in may explain part the small (but reproducible) increase in phosphopeptides. All the mitogen treatments stimulated the phos- cellular phosphotyrosine levels in these cells EGF (Table I). treat- phorylation of 80K at these same sites and at novel sites resulting ment resulted in an increase in p42 but not p36 phosphorylation. in the appearance of phosphopeptides 1, 3 and 5 (Figure 3, see Furthermore, no increase in 80K phosphorylation was observed schematic diagram in Figure 4). The mitogen-stimulated ap- suggesting that EGF treatment does not activate protein kinase pearance of the minor phosphopeptides, which migrated between C in these cells. peptides 7 and 11 and between peptides 8 and 10, was not reproducible (see Figure 4). Phosphorylation of 80K in mitogen-treated cells To examine the difference between the acidic and basic forms To obtain a quantitative estimate of the extent of 80K phos- of 80K, the phosphoproteins from untreated of gels mitogen- phorylation under various conditions, non-alkali treated were treated cells were excised in sections as indicated in gels Figure 2b, examined (Figure 2). It can be seen that 80K from mitogen-treated and subjected to peptide map analysis (Figure 4). Overall tryptic cells contained more acidic forms than that from untreated cells it was found that forms of 80K with similar from pI cells treated and that the most acidic of these tended to migrate more slowly in different ways had similar phosphopeptide patterns. For ex- than the most basic in the second dimension (Figure 2b). Up to ample, the most basic form of 80K from bombesin-treated cells 2891 et al. C.M.Isacke a b A B A 2,1 6 54 3 2 1 Bombesin Control C D 7C6,543 D 654 PDGF TPA -i Fig. 2. The 80K in two-dimensional from Swiss 3T3 cells. Two-dimensional of Swiss 3T3 cells were as region gels (a) gels [32P]orthophosphate-labeled described in 1 that the were dried before The mol. wt acidic of these 80K and Figure except gels fixing. nigher regions gels including surrounding proteins are shown. No 10 1 50 nM. time was 2 h with an screen. The same (A) addition, (B) bombesin, nM; (C) PDGF, nM; (D) TPA, Exposure intensifying (b) as shown in were and the 80K was the In the sections autoradiograms (a) enlarged region containing aligned vertically using addition, surrounding proteins. that were excised and subjected to in 4 are indicated numbers. tryptic peptide mapping Figure by a similar to the most basic form murine did not (B 1) had phosphopeptide pattern precipitated from cells [35S]methionine-labeled While of the with 80K from TPA-treated Swiss 3T3 in two- of 80K from untreated cells co-migrate cells (Control 1). many forms of and were in all the isoelectric dimensional gels phosphopeptides present (J.R.Woodgett J.Meisenhelder, unpublished 80K, most basic form of 80K some differences were noted. The results). (Con- 3 and whereas more trol 1 and B lacked 1) phosphopeptides 1, 5, Additional that bombesin activates kinase C evidence protein of these three acidic forms of the had a abundance protein greater forms of 80K a loss On toward the most acidic The activation of kinase C has been peptides. moving protein measured by a number of 8 and of 9 and 10 and 11 of indirect stimulation of 80K peptides (B 6) finally peptides assays including phosphorylation 6, was found. The data shown in 4 confirm the (TPA 7) and a of Figure (see above) decrease to its [1251]EGF binding receptor. in when the whole observation made Figure that Here we examined the in 3, namely, group [1251]EGF with binding capacity parallel of 80K are examined an increase in the amount of acidic proteins the and I and mitogenic phosphorylation asssays (Table Figure forms results in an increase in the amounts of 1). Dishes of cells were incubated for 60 with tryptic phospho- min and mitogens 3 and 5. Bombesin treatment resulted in a less com- peptides 1, [1251]EGF at 370C and the amount of radioactivity associated with conversion of basic to acidic forms of 80K than either PDGF the cells plete after this period was measured In (Table I). agreement or tumor treatments 2a and and has accor- with promoter (Figure previous reports all the mitogens tested caused a b) decrease less of 3 and 5 in EGF dingly peptides 1, binding (Brown et al., Sinnett-Smith (Figure 3). 1979; and It remained that 80K kinase C possible Rozengurt, 1985; Brown et Bombesin has represented protein al., 1984). been which is to have a mol. wt of 80 kd and be to be itself, reported reported as effective as PDGF in stimulating phospho- phosphorylated in intact cells et This inositide breakdown (Fry al., 1985). (Brown et in possibility al., 1984). However, our ex- was excluded that kinase C immuno- bombesin by demonstrating protein periments does not as an activator appear potent of 2892 Bombesin- and GRP-induced events phosphorylation *13 011 12 *X 4p8 40 4809 0.3 CONTROL BOMBESIN C D *13 p12 1-4 *7 4C . 6 _4 5 _.Je PDG TPA Fig. 3. Tryptic phosphopeptide maps and phosphoamino acid analysis of 80K from mitogen-treated Swiss 3T3 cells. The entire 80K area was excised from the two-dimensional in preparative gels shown Figure 2a and subjected to trypsin digestion and separation in two dimensions as described in Materials and methods. In each case the origin is marked (arrowhead). The peptide numbers correspond to those used in the schematic diagram in Figure 4. Electrophoresis on 100 cellulose was in the 1 thin-layer plates performed first dimension at pH 8.9 for 20 min at kV with the anode on the left. For each sample, 80K Am from 2-4 gels was pooled. Cerenkov c.p.m. in analysed each case were: (A) untreated cells, 1080 c.p.m.; (B) bombesin (10 nM), 420 c.p.m.; (C) PDGF (1 nM), 1330 c.p.m.; (D) TPA (50 nM), 1050 c.p.m. Inset: 1/8th of each 80K sample described above was subjected to phosphoamino acid analysis prior to trypsin digestion. The position of phosphoserine phosphothreonine (T) and (S), phosphotyrosine (Y) are indicated. Exposure time with an intensifying screen was 5 days. protein kinase C as either PDGF or TPA, based on the degree we examined the of in Swiss 3T3 cells phosphorylation pp6Oc-src of inhibition of EGF binding, as well as the lower level of p42 of in to treatments. The incubation response mitogen purified phosphorylation (Figure 1) and the fewer acidic forms of 80K kinase C with and in vitro or the protein pp6Oc-src [,y-32P]ATP generated (Figure 4; see Discussion). in both result in treatment of cells with tumor promoters vivo Finally, as neither the 80K phosphorylation nor EGF binding on a novel event et phosphorylation pp6Oc-src (Gould al, 1985; assays direct evidence for kinase C of 3T3 for 10 provide protein activation, Gentry et al, 1986). Treatment Swiss cells min 2893 C.M.Isacke et al. _13 *13 @13 13 _12 11 _ 12 _11 _10 1 11 11 12 8 a _ 9 4M8 _lm~ _7 ae 1pI* TPA 6 Contol i II _13 w 13 @13 _ 12 Z12 . 11 _011 40 11l _ _ 11 . 9 ; 8 8 9 12* 5_ B2 3 ' 9' I* _2 85 A B4 2 1 3331 B ; Fig. 4. Tryptic phosphopeptide maps of the acidic and basic forms of 80K from bombesin and 3T3 cells. Forms of 80K TPA, untreated Swiss with different isoelectric points were excised form preparative in 2b. gels as indicated Figure Trypsin digestion and mapping was as described in Materials and methods and in Figure 3. Maps from the most basic region of 80K from control cells (Control 1), five regions from bombesin-treated cells ranging from basic to acidic (B 1, B 2, B 3, B 4 and B 6) and three regions from TPA-treated cells (TPA 5, TPA 6 and TPA 7) are shown. For each sample 80K regions from six preparative two-dimensional gels were pooled. Cerenkov c.p.m. analysed and exposure time with an intensifying screen were: control 1, 400 c.p.m., 7 days; B 1, 210 7 B B B B c.p.m., days; 2, 620 c.p.m., 3 days; 3, 780 c.p.m., 3 days; 4, 1190 c.p.m., 3 days; 6, 430 c.p.m., 7 days; TPA 5, 1000 c.p.m., 3 TPA 1080 3 TPA 350 7 A of of is days; 6, c.p.m., days; 7, c.p.m., days. schematic diagram the tryptic phosphopeptides 80K shown. PDBu not PDGF or bombesin presence of ligand (Frackelton et al., 1984) or by Western blot- with either TPA, (data shown), stimulation of of on a ting of whole cell extracts following treatment with ligand (Zip- caused a large phosphorylation pp6oc-src residue to serine 12 in chicken pel et al., 1986). Although the bombesin receptor has not been equivalent pp6Oc-src (Figure 5), which gives rise to peptide 4. The other two characterized at the molecular level, Swiss 3T3 cells contain major pp60c-src observed in treated and untreated cells are enough bombesin receptors (- 100 000) for us to attempt to iden- phosphopeptides pep- tide which contains serine and which contains tify a protein-tyrosine kinase associated with its 1, 17, peptide 6, activity recep- Gould et et tor in this way. tyrosine 527 (Figure 5; al., 1985; Cooper al., 1986). The increased of serine 12 in To examine whether bombesin or GRP stimulate tyrosine phos- phosphorylation pp6Yc-src provides that bombesin treatment of Swiss 3T3 cells phorylation of any Swiss 3T3 cell membrane proteins, a crude further evidence of kinase C. EGF treat- membrane preparation was treated with bombesin, PDGF, or causes activation protein By contrast, in increase in EGF, incubated with and then ment did not result any pp6oc-src phosphorylation [,y-32P]ATP immunoprecipitated on 12 not that in these with anti-phosphotyrosine Ig. The EGF and PDGF receptors were serine (data shown) again demonstrating cells EGF does not activate kinase C. identified as 175 kd and 185 kd protein phosphoproteins respectively. In the described the PDGF used was a In bombesin-treated there was no detectable in experiments here, par- samples increase All these have been the phosphorylation of any membrane proteins (data not shown), tially pure preparation. experiments repeated at times with PDGF et and no different pure (Cooper al., 1982; C.M.I. phosphoprotein was specifically precipitated with K.L.G. In no case was dif- anti-phosphotyrosine Ig (Figure and unpublished 6a). We also analysed extracts observations). any ference in noted between the two PDGF of quiescent Swiss 3T3 cells treated with PDGF, EGF, response preparations. GRP or bombesin for 10-30 min by Western blotting with anti-phos- Detection with of growth factor receptors anti-phosphotyrosine photyrosine Ig. The 185 kd PDGF receptor was readily detected, antibodies in addition to of proteins 130, 70 and 33 kd (marked with ar- The failure to detect an increase in the level of phosphotyrosine rowheads in Figure 6), which were specific to PDGF-treated in total cellular or in known kinase proteins protein-tyrosine cells. The 175 kd EGF receptor was detectable in EGF-treated substrates does not the that the necessarily preclude possibility cells, but less apparent than the PDGF receptor. A protein of bombesin has intrinsic kinase receptor protein-tyrosine activity, 48 kd was in preferentially found EGF-treated cells (marked by since the bombesin have different to receptor might specificities arrowhead in In Figure 6). all the mitogen-treated samples pro- that of other known receptor protein-tyrosine kinases. Growth teins of 38 and 90 kd were observed, which were absent or less factor receptors, such as the PDGF which have receptor, intense in control samples. However, there were no striking bands associated protein-tyrosine kinase are on activity phosphorylated which were to extracts of unique bombesin- or GRP-treated cells tyrosine in the presence of their ligand. None of the techniques (Figure 6b). employed so far were designed specifically to detect autophos- of In phorylation the putative bombesin receptor. the absence Discussion of of specific anti-receptor antibodies, autophosphorylation growth factor receptors can be detected using anti-phospho- In examining phosphorylation events induced by bombesin and tyrosine antibodies either by immunoprecipitation of membrane GRP treatment of Swiss 3T3 cells, the most obvious changes are preparations phosphorylated in vitro by [-y-32P]ATP in the those which can be attributed to the activation of protein kinase 2894 Bombesin- and GRP-induced phosphorylation events CONTROL B BOMBESIN a- 1* 40 o. C PDGF D TPA A A~~~~~~~~~~~~~~~~~~~~~ A L- Fig. 5. Tryptic phosphopeptide maps of pp6O'-src from mitogen-treated Swiss 3T3 cells. pp6t-src was isolated from treated or untreated Swiss 3T3 cells by immunoprecipitation with MAb 327 (Lipsich et al., 1983), subjected to tryptic digestion and separated in two dimensions as described by Gould al. (1985). et In each case the origin is marked (arrowhead). Cells had been labeled with 2.5 mCi/ml [32P]orthophosphate for 18 h and treated for 10 140 nM 4-t- min with phorbol, (B) 10 nM bombesin, (C) 1 nM PDGF or (D) 75 nM TPA. Approximately 200 Cerenkov c.p.m. were analysed in each case. Exposure time an with screen was 5 intensifying days. C. Tumor which are to exert their confirmed that PDGF treatment, in addition to stimulating promoters, thought mitogenic effects to and kinase C activates in by binding activating protein (reviewed tyrosine phosphorylation, protein kinase C Swiss 3T3 in induce a number of cellular cells. By contrast we found no evidence that EGF induces stimula- Nishizuka, 1984; Ashendel, 1985), the indirect inhibition of EGF to its tion of kinase C. EGF events, including binding protein treatment failed to increase serine cell surface et Sinnett-Smith and 12 of receptor (Brown al., 1979; phosphorylation (data not shown) and does not pp6c-src and the increased of 80K stimulate 80K et Bishop Rozengurt, 1985) phosphorylation phosphorylation (Rozengurt al., 1983; et Blackshear et Rod- et Blackshear et (Rozengurt al., 1983; al., 1985, 1986; al., 1985; al., 1985; Figure 1). We routinely and Here we examined the ef- observed that neither bombesin nor GRP was as an efficient ac- riguez-Pena Rozengurt, 1986). fect of bombesin and GRP on these cellular events and found tivator of kinase C as the tumor GRP protein promoters, although that both criteria the bombesin activated kinase was reproducibly slightly more effective than bombesin. Tumor by peptides protein C 1 and thus the results of be more because are not as readily (Table I; Figures 2), confirming promoters may potent they Zachary et al. (1986). In addition, we employed a more direct metabolized as the DAG endogenously produced in response to for the activation of kinase the stimula- bombesin and GRP. assay protein C, namely Our of of ex- tion of serine 12 of et investigation the group 80 kd phosphoproteins phosphorylation pp6oc-src (Gould al., and showed that bombesin was able to induce this tends the work of others. Blackshear et al. (1985, 1986) 1985) phos- phorylation event (Figure 5). In the course of these studies we demonstrated that 80K from tumor PDGF or promoter, synthetic 2895 et al. C.M.Isacke a CEPB C B P E w - -_ *200 Il- iW, .1916 oil -'s. 116- i *97 - _ *68 681F 30* .30 with antibodies. For each a membrane from 6. Detection of factor anti-phosphotyrosine (a) sample crude preparation about 1 x l07 Fig. growth receptors 10 mM and incubated for 15 min at with to a concentration of 100 nM EGF Swiss 3T3 cells was resuspended in 20 Hepes (pH 7.4) 4°C mitogens give Al 1 or an volume of 1 mM acetic acid containing 1 mg/ml BSA as control (C) and then for (E), 100 nM PDGF (P), zM bombesin (B) equivalent (10 1l) as described in Materials and methods. The membranes were solubilized in RIPA buffer and immunoprecipitated 10 min at 30°C with 50 zICi [-y-32P]ATP antibodies as described in Materials and methods. One-half of each sample was resolved on a 10% SDS-polyacrylamide gel. with anti-phosphotyrosine at was 2 Swiss 3T3 cells in 35 mm dishes were treated with 10 nM bombesin (B), 1 nM Exposure time with intensifying screen -70°C days. (b) Quiescent 5 nM for or left untreated and then in SDS buffer. One-tenth of each sample was resolved on a 10% SDS- PDGF (P), EGF (E) 15 min (C) lysed sample immunoblotted as described in Materials and methods. time was 2 Mol. wt markers were (200 kd), 3- polyacrylamide gel and Exposure days. myosin bovine serum albumin ovalbumin kd) and carbonic anhydrase (30 kd). Arrowheads indicate galactosidase (116 kd), phosphorylase (97 kd), (68 kd), (43 proteins referred to in the text. on DAG-treated 3T3 cells was phosphorylated predominantly bands. to 13 were in pooled 80K Up phosphopeptides present serine residues, and resolved into several charge species. preparations. Because of the possibility of incomplete tryptic that like tumor Rozengurt and colleagues showed bombesin, pro- digestion, it is unclear exactly how many phosphorylation sites et moters, increased 80K phosphorylation (Zachary al., 1986) there are. If the charge isomers represent the addition of single and is to mediate this directly that protein kinase C very likely phosphate residues, then there can be as many as seven phos- 1986). By phosphoamino acid (Rodriguez-Pena and Rozengurt, phates per 80K molecule, implying there are at least seven distinct we found that 80K was analysis and tryptic peptide mapping phosphorylation sites. Some differences in tryptic phospho- on residues. Mitogen basally phosphorylated serine and threonine peptides content between the isoforms was evident in going from treatment caused additional phosphorylation on serine residues, basic to acidic, but there was no evidence that these phos- which in to more acidic forms. Seven resulted the conversion phorylations must occur in a strictly ordered sequence. Thus the isoelectric forms of resolved as discrete vertical 80K could be most basic form of 80K, which probably contains a single 2896 Bombesin- and GRP-induced phosphorylation events phosphate, can be phosphorylated at one of many sites. These their and induce distinct receptors yet phosphorylation responses results are consistent with there being a single 80K protein. that there are effector available implies multiple signal pathways The PDGF and EGF receptors have intrinsic ligand-stimulated for the kinases. receptor protein-tyrosine protein-tyrosine kinase activities. The fact that the response of To understand the role of bombesin and related peptides in Swiss 3T3 cells to bombesin has similarities to both the EGF an is whether the growth control, important question bombesin and PDGF responses, raises the question of whether the on Swiss 3T3 is the same bombesin cells as that on small receptor cell lung receptor might likewise have protein-tyrosine kinase activity. carcinoma A of We cells. the bombesin comparison and bombesin- failed to detect a marked increase in tyrosine related to these cell lines phosphorylation that the same peptide binding suggests of cellular proteins following bombesin treatment of is utilized in both cases quiescent and receptor (Zachary Rozengurt, 1985; cells, but the methods we used to detect such a stimulation have et or an of Moody al., 1985). [1251]GRP analogue bombesin con- limitations. For instance, only a minor increase in total phospho- an iodinatable taining tyrosine residue, [Tyr4-125I]bombesin, tyrosine levels is detected in cells following EGF treatment bind to a class of = unless single high affinity sites 0.5 In (KD nM). the cells have unusually high levels of EGF receptors such both cell this is with as bombesin-related types binding displaceable A431 cells (Hunter and Cooper, 1981). Thus even though which have Swiss peptides mitogenic activity, e.g., bombesin, (Tyr4) 3T3 cells possess about 85 000-100 000 EGF receptors bombesin and but not (Brown GRP, inactive such as vaso- by peptides et al., 1984; Zachary et al., 1986) and are mitogenically vasoactive intestinal respon- pressin, or substance P. an peptide Thus, sive to EGF (Table I), we did not observe a significant of the increase mechanism which the understanding by bombesin/GRP in phosphotyrosine levels following EGF treatment. on Two-dimen- receptor Swiss 3T3 cells transmits its mitogenic signal may sional gel analysis phosphoproteins of for well an phosphotyrosine- provide insight into the role of the bombesin-related pep- containing proteins also has shortcomings, which we tides in the autocrine have stimulation of small cell lung carcinomas. discussed elsewhere (Cooper and Hunter, 1983). For example, despite relatively the large increase in total phosphotyrosine in Materials and methods PDGF-treated cells, the only phosphotyrosine-containing proteins Materials detected by this technique are p42 and p36 (Cooper et al., 1982). PDGF 0.4% was a from ( pure) gift Elaine Raines (University of Washington, We were also unable to obtain direct evidence that the bom- Raines and Seattle, WA; EGF was obtained from Ross, 1982). S.Potter (The besin/GRP receptor has a ligand-stimulated protein-tyrosine Salk and Institute; Savage Cohen, 1972). 4-13-Phorbol, 4-3-phorbol-12,13-di- kinase activity, by using anti-phosphotyrosine and antibodies butyrate 12-O-tetradecanoyl-phorbol-13-acetate either (PDBu) (TPA) were obtain- ed from Chemicals. Sigma Bombesin and GRP were a for immunoprecipitation gift from Jean Rivier of in vitro phosphorylated (The membrane Salk Institute; Rivier and Brown, 1978). Peptides were dissolved in water or preparations or for Western blotting of whole-cell extracts, techni- Dulbecco-Vogt modified Eagle's medium (DMEM) and stored 1 as mM stock ques which allowed detection of the PDGF receptor. This con- solutions at -20°C. Concentrations of mitogens used in each assay are given trasts with a recent study by Comoglio and co-workers in the (Cirillo figure legends. et al., 1986), where a 105 kd protein was observed by Western Cells blotting extracts bombesin-treated of Swiss 3T3 cells with bombesin-responsive Swiss 3T3 cell clone was obtained from Enrique Rozengurt anti-phosphotyrosine Ig. These workers were also able to im- (ICRF, London), and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). munoprecipitate Unless otherwise stated confluent, quiescent cells a protein of this size, whose were prepared phosphorylation for x assays by seeding 2.5 101 cells into a 35 mm dish in DMEM on tyrosine supplemented in vitro was stimulated by bombesin. Their results with 10% FBS and culturing for 6-7 days. For metabolic labeling, the medium suggest that the bombesin receptor is a protein of 105 kd with was then aspirated and the cells cultured for a further 18 h in 1 ml of phosphate- ligand-stimulated protein-tyrosine kinase activity. Our failure to free DMEM containing 5% (v/v) DMEM and 2 mCi of [32P]orthophosphate detect such a protein may be attributable to the (ICN). Mitogens were added for 15 min prior to anti-phospho- lysis. A crude membrane preparation was made from tyrosine antibody preparation Swiss 3T3 cells as follows: we used, since there is documented cells - ( 80% confluent) were scraped from mm 150 dishes at 4°C in 2 ml of variability in the affinity of anti-phosphotyrosine antibodies 5 mM Hepes (pH 7.4), 2 mM MgCl2, 5 mM 2-mercaptoethanol, 0.1% aprotinin, towards individual phosphotyrosine-containing proteins. 0.1 mM 4 PMSF, 4 SBTI, leupeptin and homogenized with 25 jug/ml ;sg/ml If the bombesin receptor has protein-tyrosine kinase strokes in activity, a Dounce homogenizer using a tight fitting pestle. The homogenate was centrifuged at 200 for 5 mins to remove remaining whole cells, our analysis of tyrosine phosphorylation events nuclei in the intact g and cell cell debris, and then at 20 000 g for 60 min. The supematant was discarded and suggests that the bombesin receptor either has a lower specific the membrane pellet stored in liquid N2. activity or else has a much more restricted substrate specificity Assays than the PDGF receptor protein-tyrosine kinase. In this respect Whole cell phosphoamino acid analysis. This was performed according to Sef- the properties of the bombesin receptor are more akin to those ton et al. (1980). of the EGF receptor, since treatment by neither factor results Two-dimensional gel electrophoresis. This was performed using the method of in elevation an in whole-cell phosphotyrosine levels. However, Garrels (1979) modified by Cooper and Hunter (1981) except that isoelectric focus- a major difference in the mechanism of action of the was bombesin ing with pH 3.5-10 ampholytes (LKB). The gels were fixed, dried and rehydrated 1 and EGF receptors is observed; while PDGF and in M KOH, incubated at 550C for 2 h, neutralized, bombesin and dried again are (Cooper and Hunter, 1981). Preparative gels for 80K were rinsed for 30 min potent activators of PI turnover in Swiss 3T3 cells (Habenicht in water with mixed-bed ion resin exchange (Amberlite) and dried without fixing. et al., 1981; Brown et al., 1984), EGF treatment of most cells, Inhibition ofEGF binding. This was to assayed according Brown et al. (1984). including Swiss 3T3 cells, does not result in the activation of Essentially dishes of cells were cultured overnight in labeling medium without protein kinase C (Bishop et al., 1985; Blackshear et al., 1985; [32P]orthophosphate and then incubated at 370C for 60 min in 1 ml DMEM con- Habenicht et al., 1981; Coughlin et al., 1985). Clearly one needs taining mitogens and 27 000 c.p.m. [125I]EGF (NEN; 175 After washing jiCi/4g). in ice-cold to identify the substrate for the PDGF receptor DMEM, the cells were extracted with 0.5 M NaOH and protein-tyrosine counted in a y-counter. kinase which when phosphorylated is responsible for the increase Measurement of [3H]thymidine incorporation. Dishes of cells were in DAG level, and determine whether this cultured for is also phosphorylated a further 24 h with 2 ml of DMEM. Mitogens together with 1 in insulin were bombesin-treated cells, or whether itg/ml bombesin activates PT turn- added for another 24 h, and the cells were labeled with 2 [methyl- isCi/ml over in another manner. The fact that PDGF, EGF and probably 3H]thymidine (6.7 Ci/mmol, NEN) for the final 6 h. Cells were then fixed with bombesin all stimulate tyrosine phosphorylation upon binding to methanol:acetic acid (3:1, v/v), washed with 10% extensively trichloroacetic acid 2897 C.M.Isacke et al. (TCA) at 4°C, rinsed with PBS and solubilized in 1 ml 0.2 M NaOH, 0.5 ml Cooper,J.A. and Hunter,T. (1981) Mol. Cell. Biol., 1, 165-178. was counted in 10 ml of aqueous scintillation fluid. Cooper,J.A. and Hunter,T. (1983) Curr. Topics Microbiol. Immunol., 107, 125- 161. Peptide mapping and phosphoamino acid analysis. 80K phosphoproteins were Mol. Cell. Cooper,J.A. and Hunter,T. (1985) Biol., 5, 3304-3309. excised from the preparative gels and then protein was subjected to phosphoamino Cooper,J.A, Bowen-Pope,D.F., Raines,E., and Hunter,T. acid analysis or tryptic peptide mapping as described by Hunter and Sefton (1980). Ross,R. (1982) Cell, 263-273. 31, The 32P-labeling of pp6(j-src in vivo and its tryptic peptide mapping have been Cooper,J.A., Sefton,B.M. and Hunter,T. (1984) previously described Mol. Cell. Biol., 4, 30-37. (Gould et al., 1985). In this experiment mitogen treatments Cooper,J.A., Gould,K.L., Cartwright,C.A. and Hunter,T. (1986) were for 10 min Science, 231, prior to lysis. 1431-1433. Western blotting. Dishes of cells were cultured for a further 24 h in DMEM alone Corps,A.N., Rees,L.H. and Brown,K.D. (1985) Biochem. J., 231, 781-784. and then treated with mitogens for 10 to 30 min before rinsing once with cold Coughlin,S.R., Lee,W.M.F., Williams,P.W., Giels,G.M. and Williams,L.T. PBS and lysing the cells in 0.3 ml boiling Laemmli sample buffer. The samples (1985) Cell, 43, 243-251. were boiled for 5 min, sheared by repeated passage through a 27 gauge needle, Cuttitta,F., Carney,D.N., Mulshine,J., Moody,T.W., Fedorko,J., Fischler,A. reboiled for 20 min and equal volumes were resolved on 10% SDS-polyacryla- and Minna,J.D. (1985) Nature, 316, 823-826. mide gels. Proteins were transferred to 0.45 nitrocellulose (Schleicher itm and Erisman,M.D., Linnoila,R.I., Hernandez,O., DiAugustine,R.P. and Lazarus,L.H. Schuell) in a BioRad apparatus using a Tris-glycine buffer containing 20% (1982) Proc. Natl. Acad. Sci. USA, 79, 2379-2383. methanol at a current of 40 V for 90 min. The blot was then blocked overnight Frackelton,A.R.,Jr, Tremble,P. and Williams,L.T. (1984) J. Biol. Chem., 259, in rinse buffer (10 mM pH 7.4, 0.15 M NaCI) containing 5% BSA Tris-HCI, 7907-7914. and 1% ovalbumin and probed for 2 h in same buffer containing the 4 Fry,M.J., Gebhardt,A., Parker,P. and Foulkes,J.G. Agg/ml (1985) EMBO J., 4, affinity-purified anti-phosphotyrosine Ig generated by the immunization of rab- 3173-3178. bits with poly[phosphotyrosine.Ala.Gly] covalently linked to keyhold limpet hemo- Garrels,J.I. (1979) J. Biol. Chem., 254, 7961-7977. cyanin (a kind gift of Mark Kamps, Salk Institute). The blot was then washed Gentry,L.E., Chaffin,K.E., Shoyab,M. and Purchio,A.F. (1986) Mol. Cell. Biol., twice in rinse buffer, once in rinse buffer containing 0.05% NP-40, and then 6, 735-738. twice more in rinse buffer for 10 min each. The blot was incubated with [1251]- Gilmore,T. and Martin,G.S. (1983) Nature, 306, 487-490. protein A for 1 h to decorate bound antibody, washed as above, dried and ex- Gould,K.L., Woodgett,J.R., Cooper,J.A., Buss,J.E., Shalloway,D. and Hunter,T. posed to film. (1985) Cell, 42, 849-857. Immunoprecipitation of phosphorylated membrane proteins. Crude membrane Habenicht,A.J., Glomset,J.A., King,W.C., Nist,C., Mitchell,C. and Ross,R. - 1 preparations (see above) from x 107 cells were resuspended in 20 of 1I (1981) J. Biol. Chem., 256, 12329-12335. 10 mM Hepes (pH 7.4), incubated for 15 min at 4°C with mitogens and then Hunter,T. and Cooper,J.A. (1981) Cell, 24, 741-752. phosphorylated in vitro for 10 min at 30°C with 50 /Ci [-y-32P]ATP Hunter,T. and Cooper,J.A. (1985) Ann. Rev. Biochem., 54, 897-930. (3000 Ci/mmol, Amersham) in 25 of 20 mM Pipes (pH 7.4), 10 mM MnCl2, Hunter,T. and Sefton,B.M. (1980) Proc. Natl. Acad. Sci. USA, 77, 1311-1315. A1 4 unlabeled ATP (final concentration). Membranes were then solubilized in Isacke,C.M., Trowbridge,I.S. and Hunter,T. (1986) Mol. Cell. ytM Biol., 6, 0.5 ml RIPA buffer (Sefton et al., 1978) containing 50 mM Tris, pH 7.4 in- 2745-2751. stead of phosphate buffer and supplemented with mM EDTA; 100 5 u1M Na3VO4; Lipsich,L.A., Lewis,A.J. and Brugge,J.S. (1983) J. Virol., 48, 352-360. 50 mM NaF. The lysate was then clarified by centrifugation at 20 000 g for 60 min McDonald,T.J., Jornvall,H., Nilsson,G., Vagne,M., Ghatei,M., Bloom,S.R. and and immunoprecipitated according to Sefton et al. (1978) using anti-phospho- Mutt,V. (1979) Biochem. Biophys. Res. Commun., 90, 227-233. tyrosine Ig affinity-purified from a rabbit antiserum raised against phosphotyrosine Moody,T.W., Pert,C.B., Gazdar,A.F., Carney,D.N. and Minna,J.D. (1981) coupled to BSA (obtained from Mark Kamps, The Salk Institute). Science, 214, 1246-1248. Moody,T.W., Carney,D.N., Cuttitta,F., Quattrocchi,K. and Minna,J.D. (1985) Autoradiography. This was performed using Kodak XAR film, which where in- Life Sci., 37, 105-113. dicated was pre-sensitized and exposed at -70°C with an intensifying screen. Nishizuka,Y. (1984) Nature, 308, 693-698. Radke,K. and Martin,G.S. (1979) Proc. Natl. Acad. Sci. USA, 76, 5212-5216. Acknowledgements Raines,E. and Ross,R. (1982) J. Biol. Chem., 257, 5154-5160. We are extremely grateful to Enrique Rozengurt for providing us with Ralston,R. and Bishop,J.M. (1985) Proc. Natl. Acad. Sci. the USA, 82, 7845-7849. bombesin-responsive clone of Swiss 3T3 cells and also communicating Rivier,C., Rivier,J.E. and Vale,W. for results (1978) Endocrinol., 102, 519-524. prior to publication, and to Paolo Comoglio for informing us of experiments Rivier,J.E. and Brown,M.R. (1978) Biochemistry, con- 17, 1766-1771. cerning the nature Rodriguez-Pena,A. and of the bombesin receptor prior to publication. We thank Jean Rozengurt,E. (1985) EMBO J., 4, 71-76. Rivier for supplying bombesin and gastrin-releasing peptide and Mark Rodriguez-Pena,A. Kamps and Rozengurt,E. (1986) EMBO J., 5, 77-83. for the affinity-purified anti-phosphotyrosine antibodies. This work was supported Rozengurt,E. and Sinnett-Smith,J. (1983) Proc. Natl. Acad. Sci. USA, 80, by Public Health Service Grant Numbers 2936-2940. CA 17096, CA 28458 and CA 39780 Rozengurt,E., from the National Cancer Institute. C.M.I. was a recipient of a fellowship from Rodriguez-Pena,M. and Smith,K.A. (1983) Proc. Natl. Acad. Sci. the Damon Runyon-Walter Winchell Cancer Fund (DRG-775). K.D.B. was USA, 80, 7244-7248. a recipient of a grant from the Nuffield Foundation and is grateful to Bob Holley Savage,C.R.,Jr and Cohen,S. (1972) J. Biol. Chem., 247, 7609-7611. at the Salk Institute for providing support and laboratory space. K.L.G. and S.J.G. Sefton,B.M., Beemon,K. and Hunter,T. (1978) J. Virol., 28, 957-971. were supported by National Institutes of Health predoctoral training grants to Sefton,B.M., Hunter,T., Beemon,K. and Eckhart,W. (1980) Cell, 20, 807-816. the University of California at San Diego. Sinnett-Smith,J.W. and Rozengurt,E. (1985) J. Cell. Physiol., 124, 81-86. Swope,S.L. and Schonbrunn,A. (1984) Proc. Natl. Acad. Sci. USA, 81, 1822-1826. References Weber,S, Zuckerman,J.E., Bostwick,D.G., Bensch,K.G., Sikic,B.I. and Raffin, Anastasi,A., Erpsamer,V. and Bucci,M. (1971) Experientia, 27, 166-167. (1985) J. Clin. T.A. Invest., 75, 306-309. Ashendel,C.L. (1985) Biochem. Biophys. Acta, 822, 219-242. Westendorf,J.M. and Schonbrunn,A. (1982) Endocrinology, 110, 352-358. Blackshear,P.J., Witters,L.A., Girard,P.R., Kuo,J.F. and Quamo,S.N. (1985) Westendorf,J.M. and Schonbrunn,A. (1983) J. Biol. Chem., 258, 7527-7535. J. Biol. Chem., 260, 13304-13315. Willey,J.C., Lechner,J.F. and Harris,C.C. (1984) Exp. Cell Res., 153, 245-248. Blackshear,P.J., Wen,L., Glynn,B.P. and Witters,L. (1986) J. Biol. Chem., 261, Zachary,I. and Rozengurt,E. (1985) Proc. Natl. Acad. Sci. USA, 82,7616-7620. 1459-1469. Zachary,I., Sinnett-Smith,J.W. and Rozengurt,E. (1986) J. Cell Biol., 102, Bishop,R., Martinez,R., Weber,M.J., Blackshear,P.J., Beatty,S., Lim,R. and 2211 -2221. Herschman,H.R. (1985) Mol. Cell. 5, 2231-2237. Biol., Zippel,R., Sturani,E., Toschi,L., Naldin,L., Alberghina,L. and Comoglio,P.M. Brown,K.D. Laurie,M.S. and (1986) J. Physiol., 71, p. 210. (1986) Biochem. Biophys. Acta, 881, 56-61. Brown,K.D., Dicker,P. and Rozengurt,E. (1979) Biochem. Biophys. Res. Commun., 86, 1037-1043. Received on 2 April 1986; 1 revised on September 1986 Brown,K.D., Blay,J., Irvine,R.F., Heslop, J.P. and Berridge,M.J. (1984) Biochem. Biophys. Res. Commun., 123, 377-384. Brown,M., Marki,W. and Rivier,J. (1980) Life Sci., 27, 125-128. Cirillo,D.M., Gaudino,G., Naldini,L. and Comoglio,P.M. (1986) Mol. Cell. Biol., in press. Collins,M.K.L. and Rozengurt,E. (1982) J. Cell. Physiol., 112, 42-50. Collins,M.K.L. and Rozengurt,E. (1984) J. Cell. Physiol., 118, 133-142.

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The EMBO Journal – Springer Journals

Published: Nov 1, 1986

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