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Bombesin and Neuromedin B Stimulate the Activation of P42mapk and P74raf-1 Via a Protein Kinase C-Independent Pathway in Rat-1 Cells

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Bombesin and Neuromedin B Stimulate the Activation of P42mapk and P74raf-1 Via a Protein Kinase C-Independent Pathway in Rat-1 Cells Oncogene (1997) 14, 2323 ± 2329 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Bombesin and neuromedin B stimulate the activation of p42mapk and p74raf-1 via a protein kinase C-independent pathway in Rat-1 cells Amanda Charlesworth and Enrique Rozengurt Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK The mechanisms by which seven transmembrane recep- (PKC)-dependent pathways have been described (Howe tors activate p42mapk/p44mapk are not well de®ned although and Marshall, 1993; Crespo et al., 1994; Seuerlein and p21ras- and protein kinase C (PKC)-dependent pathways Rozengurt, 1995; Seuerlein et al., 1995, 1996; van have been implicated, typically for Gi- and Gq-coupled Biesen et al., 1996) typically for pertussis toxin (PTx)- receptors, respectively. Here, we demonstrate that in sensitive Gi and PTx-insensitive Gq-coupled receptors Rat-1 cells transfected with the Gq-coupled bombesin/ respectively (Hawes et al., 1995). gastrin releasing peptide receptor, bombesin stimulated The peptides of the bombesin family including gastrin activation of p42mapk that was not inhibited by the speci®c releasing peptide (GRP) are potent mitogens for a PKC inhibitor GF 109203X or by down regulation of variety of cells including Swiss 3T3 cells (Rozengurt phorbol ester-sensitive PKC isoforms. In addition, and Sinnett-Smith, 1983; Zachary and Rozengurt, 1985) bombesin rapidly stimulated p74raf-1 activity that was and Rat-1 cells (Charlesworth et al., 1996) and have been also independent of PKC activity and insensitive to implicated as autocrine growth factors for small cell lung inhibition by pertussis toxin. Furthermore, addition of cancer (Cuttitta et al., 1985; Carney et al., 1987; Sethi et neuromedin B to Rat-1 cells transfected with the al., 1991). Bombesin binds to a speci®c Gq-coupled seven neuromedin B preferring receptor also activated p42mapk transmembrane domain receptor (Zachary and Rozen- and p74raf-1 in a PKC-independent and pertussis toxin- gurt, 1985, 1987; Coer et al., 1990; Battey et al., 1991; insensitive manner. Finally we show that addition of Oermans et al., 1994) and elicits rapid phospholipase C bombesin to Rat-1 cells stimulated the GTP loading of mediated hydrolysis of polyphosphoinositides leading to p21ras. Our results reveal a novel PKC-independent Ca2+ mobilisation from intracellular stores and activa- pathway in the action of Gq-coupled receptors and stress tion of PKC (Rozengurt, 1992). Addition of bombesin to the importance of cell context in de®ning the signal Swiss 3T3 cells also induces rapid activation of p42mapk transduction pathway(s) that link speci®c receptors to and p44mapk (Withers et al., 1995) that is completely the activation of the mitogen-activated protein kinase dependent on PKC (Pang et al., 1993; Seuerlein et al., cascade. 1996) and does not involve p21ras (Satoh et al., 1990; Mitchell et al., 1995), or p74raf-1 (Mitchell et al., 1995; ras mapk Keywords: p21 ;Gq; neuropeptide; MAP kinases; Seuerlein et al., 1996). Thus, the mechanism for p42 / signal transduction p44mapk activation by bombesin in Swiss 3T3 cells is consistent with the model for general Gq-coupled receptor activation. However, some studies have indicated that p21ras could be involved in p42mapk/ mapk Introduction p44 activation by Gq-coupled receptors (Crespo et al., 1994; Koch et al., 1994; Mattingley et al., 1994) and The mitogen-activated protein kinases (MAPKs) are a currently there is increasing recognition of the impor- family of evolutionary conserved serine/threonine tance of the cellular context in which the MAPK kinases that are activated by a range of extracellular pathway is activated (Dikic et al., 1996; van Biesen et signals (Davis, 1993; Marshall, 1995). The two best al., 1996; Wan et al., 1996). characterised isoforms p42mapk/ERK-2 and p44mapk/ In the present study we examined the mechanism of ERK-1 are directly activated by phosphorylation on p42mapk activation by bombesin in Rat-1 cells stably speci®c tyrosine and threonine residues by the dual transfected with the bombesin/GRP preferring receptor speci®city MAPK kinase (or MEK) of which at least (Charlesworth et al., 1996). Our results reveal a novel two isoforms have been identi®ed in mammalian cells PKC-independent pathway in the action of Gq-coupled (Payne et al., 1991; Crews et al., 1992; Wu et al., 1993). receptors and stress the importance of cell context in Several pathways leading to MEK activation have been de®ning the signal transduction pathways that link demonstrated. Tyrosine kinase receptors induce p42mapk speci®c receptors to the activation of the MAP kinase and p44mapk activation via SOS-mediated accumulation cascade. of p21ras-GTP, which then activates a kinase cascade comprising p74raf-1, MEK and p42mapk/p44mapk (Davis, 1993; Marshall, 1995). The mechanisms by which mitogenic guanine nucleotide binding protein (G Results and Discussion protein) coupled receptors activate p42mapk and p44mapk Bombesin stimulates p42mapk activity in Rat-1 cells are less well de®ned although p21ras- and protein kinase transfected with the bombesin/GRP receptor mapk Correspondence: E Rozengurt To examine bombesin stimulation of p42 activity in Received 1 November 1996; revised 29 January 1997; accepted Rat-1 cells transfected with the bombesin/GRP 31 January 1997 receptor, quiescent BOR 15 cells were treated with PKC-independent Gq activation of MAPK pathway A Charlesworth and E Rozengurt 2324 increasing concentrations of this agonist for 5 min. down regulation of phorbol ester-sensitive isoforms of Lysates from stimulated and unstimulated cells were PKC by prolonged treatment with 800 nM PDB, and immunoprecipitated with antibodies against p42mapk by using the speci®c PKC inhibitor GF 109203X. This and then the activity of this enzyme was measured by compound is a bisindolylmaleimide which selectively an immune-complex kinase assay, using myelin basic inhibits diacylglycerol-regulated PKC isoforms by protein (MBP) peptide as substrate. As shown in competing with ATP for the ATP binding site Figure 1 (left), bombesin induced a concentration (Toullec et al., 1991; Yeo and Exton, 1995). The dependent increase in p42mapk activity, the half results presented in Figure 1 (right) show that phorbol maximum and maximum concentration being 0.3 and ester-mediated p42mapk activation was signi®cantly 10 nM respectively. The maximum stimulation of attenuated by GF 109203X (66% inhibition) and p42mapk activity induced by bombesin (15-fold over completely abolished by chronic pretreatment with baseline levels) was comparable to the increase in PDB. As expected, EGF-induced p42mapk activation in p42mapk activity induced by epidermal growth factor Rat-1 cells was not signi®cantly inhibited by PKC (EGF) (Figure 1, inset). In contrast, addition of inhibition and only slightly by PKC down regulation. phorbol 12,13-dibutyrate (PDB) to parallel cultures In contrast to previous results with Swiss 3T3 cells, activated p42mapk to a level that was only 20 ± 30% of bombesin-induced p42mapk activation was not signifi- that induced by bombesin or EGF (Figure 1, inset). cantly inhibited by either GF 109203X or pretreatment Thus, bombesin stimulated a robust p42mapk activation with PDB (Figure 1, right). Thus, the results in Figure in Rat-1 cells transfected with the bombesin/GRP 1 demonstrate that bombesin induces a strong receptor. stimulation of p42mapk activity in Rat-1 cells trans- In view of the role of phorbol ester-sensitive fected with the bombesin GRP receptor through a mapk mapk isoforms of PKC in Gq-mediated p42 and p44 PKC-independent pathway. activation (Pang et al., 1993; Hawes et al., 1995; Seuerlein et al., 1996), it was important to assess the Bombesin stimulates p74raf-1 activity independently of contribution of these PKCs to the p42mapk response PKC in Rat-1 cells induced by bombesin in the bombesin/GRP receptor transfected Rat-1 cells. This was examined both by G protein-coupled receptors activate p42mapk and p44mapk through p74raf-1-dependent and independent pathways (Russell et al., 1994; Hawes et al., 1995; Mitchell et al., 1995; Seuerlein et al., 1995). Consequently, we examined whether bombesin stimu- lates p74raf-1 activity in Rat-1 cells transfected with the bombesin/GRP receptor. This was achieved using a sensitive assay in which the activation of immunopre- cipitated p74raf-1 kinase from bombesin-stimulated cells Figure 1 (Left) Bombesin-induced increase in p42mapk activity. BOR 15 cells in 100 mm dishes were washed twice in DMEM and incubated in 5 ml DMEM with increasing concentrations of bombesin for 5 min at 378C. Cells were lysed and p42mapk was immunoprecipitated with polyclonal anti-p42mapk antibodies. p42mapk activity was measured by an in vitro kinase assay using MBP peptide as a substrate as described in Materials and methods. Results shown are the mean of two independent experiments that were performed in duplicate. (Inset) p42mapk Figure 2 (a) Time course of bombesin-induced p74raf-1 activity. activation induced by various factors. BOR 15 cells in 100 mm BOR 15 cells in 100 mm dishes were washed twice in DMEM and dishes were washed twice with DMEM and incubated in 5 ml incubated in 5 ml DMEM with 1 nM bombesin for increasing raf-1 DMEM without (7) or with 1 nM bombesin (B), 200 nM PDB times at 378C. Cells were lysed and p74 was immunoprecipi- (P) or 5 ng/ml EGF (E) for 5 min at 378C. p42mapk was tated with polyclonal C12 anti-p74raf-1 antibody. p74raf-1 activity immunoprecipitated followed by an in vitro kinase reaction as was measured by a two stage in vitro immunecomplex kinase above. Results shown are from one experiment performed in assay as described in Materials and methods. Results shown are duplicate. Similar results were obtained in two independent the mean of two independent experiments performed in duplicate. experiments (Right) Eect of PKC inhibition and down (b) Eect of PKC inhibition and down regulation on p74raf-1 regulation on bombesin-stimulated p42mapk activity.
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