Oncogene (1997) 15, 53 ± 61  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Association of Grb2 with Sos and Ras with Raf-1 upon gamma irradiation of breast cancer cells

Simeng Suy1, Wayne B Anderson4, Paul Dent5,6, Esther Chang2 and Usha Kasid1,3

Departments of 1Radiation Medicine, 2Otolaryngology-Head and Neck Surgery and 3Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, Washington DC 20007; 4Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; 5Howard Hughes Medical Institute, Departments of Medicine and Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA

Raf-1 protein serine/threonine kinase has been impli- Ras (Smith et al., 1986; Wood et al., 1992; Koide et al., cated in growth and damage-responsive signal transduc- 1993; McCormick, 1993; Schaap et al., 1993). Reports tion pathways. Several reports indicate an important role from several laboratories suggest that catalytic activa- of Ras protein in the growth factor-induced activation of tion of Raf-1 requires its phosphorylation or acidic Raf-1. Here we investigated the possible involvement of substitutions of tyrosines at positions 340 and 341, and Ras in ionizing radiation-induced activation of Raf-1. association with Ras (Fabian et al., 1993, 1994; Finney Irradiation of MDA-MB 231 human breast cancer cells et al., 1993; Koide et al., 1993; Moodie et al., 1993; caused an increase in GTP-binding and hydrolysis on Van Aelst et al., 1993; Vojtek et al., 1993; Warne et al., Ras, and co-immunoprecipitations of endogenous Grb2 1993; Hallberg et al., 1994). Protein tyrosine phosphor- with Sos and Raf-1 with Ras. An increase in the level of ylationofRaf-1hasbeendemonstratedincells membrane-bound Raf-1, and tyrosine-phosphorylation of overexpressing Ras and/or activated Src (Williams et Raf-1 were observed after irradiation. Consistent with al., 1992; Fabian et al., 1993; Marais et al., 1995; these changes, irradiation of cells stimulated the catalytic Jelinek et al., 1996). In hematopoietic cells, tyrosine activity of Raf-1. Finally, radiation treatment of breast phosphorylation of Raf-1 has been reported in cancer cells led to an increase in the phosphorylation and response to external stimuli in the absence of over- activity of the mitogen-activated protein kinase. Based expressed oncogenes (Carroll et al., 1990; Thompson et on these biochemical modi®cations in vivo, we conclude al., 1991; Reed et al., 1993; Turner et al., 1993). that Raf-1 functions as an e€ector of Ras in the Furthermore, inactivation of puri®ed Raf-1 from ras radiation-responsive signal transduction pathway leading expressing SF9 cells by the tyrosine-speci®c phospha- to the activities of Raf-1 and mitogen-activated protein tase PTP-1B indicates that Ras-induced activation of kinase. Raf-1 is dependent on tyrosine phosphorylation (Dent et al., 1995; Jelinek et al., 1996). The interaction of Ras Keywords: ionizing radiation; Grb2; Sos; Ras; Raf; and Raf has been shown in vitro, in the yeast two- MAPK; breast cancer hybrid system, and in mammalian cells (Koide et al., 1993; Moodie et al., 1993; Traverse et al., 1993; Zhang et al., 1993). The binding of Ras and Raf is dependent on Ras being in the GTP-bound state, and occurs Introduction through the e€ector site of Ras and the CR1 conserved region on Raf (Pumiglia et al., 1995). In addition, The cellular response to external stimulus involves membrane translocation of Raf-1 is the result of its transduction of a series of biochemical and molecular binding to Ras (Schaap et al., 1993; Stokoe et al., 1994; signals which regulates cell growth and proliferation. Fabian et al., 1994; Marais et al., 1995). Ras ± Raf The Raf-1 and Ras proteins are among the predomi- interaction and activation of Raf-1 lead to the nant components of mitogenic signal transduction activation of the mitogen-activated serine/threonine pathways (Rapp, 1991; Roberts, 1992; Satoh et al., protein kinase (MAPK) or extracellular signal-regu- 1992; McCormick, 1993; Crews and Erikson, 1993). lated kinase (Erk) through an activation of mitogen- Ras protein is a membrane-localized, guanine nucleo- activated protein kinase kinase (MAPK kinase, MKK) tide-binding protein that is biologically active when also referred to as MAP/Erk kinase (MEK) (Dent et bound to GTP and inactive when bound to GDP al., 1992; Howe et al., 1992; Kyriakis et al., 1992; (Downward, 1992). Growth factors that activate Robbins et al., 1992; Hipskind et al., 1994; Macdonald protein tyrosine kinases (PTKs) stimulate the nucleo- et al., 1993). MAPKs translocate to the nucleus upon tide exchange rate on Ras and thus regulate its activity activation, and play an important role in the activation (Bollag and McCormick, 1991; Li et al., 1992; Zhang et of transcription factors (Devary et al., 1992; Marais et al., 1992; Aronheim et al., 1994). al., 1993; Radler-Pohl et al., 1993; Cowley et al., 1994). Raf-1, a cytosolic protein serine/threonine kinase, We and others have shown that treatment of has been shown to function downstream of PTK(s) and mammalian cells with ionizing radiation (IR) leads to the activation of PTK(s) and tyrosine phosphorylation of several proteins (Uckun et al., 1993; Kasid et al., Correspondence: U Kasid. 1996). Ionizing radiation has been shown to increase 6Present address: Department of Radiation Oncology, Medical the DNA binding activity of transcription factors College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA. (Hallahan et al., 1993; UK, unpublished data). We Received 26 November 1996; revised 19 March 1997; accepted 20 have demonstrated that IR exposure of laryngeal March 1997 squamous carcinoma cells causes tyrosine phosphor- Radiation activates the Ras signaling pathway SSuyet al 54 ylation and activation of Raf-1 (Kasid et al., 1996). Grb2 was observed (Figure 2, top). When the same While, the interaction of Ras and Raf has been blot was reprobed with anti-Grb2 antibody, no change established in response to mitogenic stimulation, the in the levels of Grb2 protein was noted after IR (Figure physiological relevance of Ras and Raf interaction in 2, bottom). Together with the data shown in Figure 1, cells exposed to IR is not known. In the present study, these observations suggest that Sos acts as an activator we have investigated the possibility of a Ras signaling of Ras in irradiated breast cancer cells. pathway stimulated by IR. The results of our experiments demonstrate that IR treatment of breast Radiation stimulates association of Raf-1 with Ras, and cancer cells results in the association of the Ras membrane-recruitment of Raf-1 in vivo nucleotide exchange factor Son of sevenless (Sos) with the SH2/SH3 adaptor Growth factor receptor-bound We then examined the possibility of the association of protein 2 (Grb2), increased level of Ras.GTP, and Raf-1 with Ras following irradiation. Whole cell binding of Raf-1 to Ras. We show that these lysates prepared at 30 min after IR were immunopre- interactions correlate with membrane-recruitment, cipitated with anti-Ras antibody (238). The Ras tyrosine phosphorylation and activation of Raf-1, followed by activation of MAP kinase.

Results ¨ GDP Radiation stimulates GTP binding of Ras in vivo Recently our laboratory has demonstrated that activa- tion of Raf-1 in head and neck squamous carcinoma cells (PCI-04A) exposed to ionizing radiation (IR)

occurs as a result of its recruitment to the membranes ¨ GTP and tyrosine phosphorylation (Kasid et al., 1996). Since Raf-1 activation has been linked to the activity of Ras in the ras transformed cells (Wartmann and Davis, 1994; Marais et al., 1995; Jelinek et al., 1996), we reasoned that IR-stimulation of Raf-1 could also be due to an UI 5' 15' 30' ML increase in the active form of Ras (Ras.GTP) in irradiated cells. To investigate this possibility, whole Post-IR Time cell extracts (WCE) of irradiated or unirradiated cells Figure 1 Radiation stimulates GTP binding of Ras in MDA-MB were immunoprecipitated with monoclonal antibody 231 cells. Cells were serum-starved for 18 h prior to irradiation (15 Gy, 114 cGy/min). At indicated times after IR, cells were against Ras (H-Ras,259). The Ras immunoprecipitates lysed and WCE (1 mg) were immunoprecipitated with agarose- were then subjected to radiolabeling with [a32P]GTP, conjugated anti-H-Ras monoclonal antibody (259). To assess and the level of bound-GTP and hydrolysis of GTP on GTP binding and hydrolysis on Ras, the immune-complexes were Ras (GTP:GDP) were determined in a TLC assay. incubated with 1 mCi of [a-32P]GTP (3000 Ci/mmol). The bound guanylnucleotides (GTP and GDP) were eluted and separated by Representative data is shown in Figure 1. IR stimulated thin layer chromatography, followed by autoradiography. The a transient increase in the level of Ras.GTP within migrations of GTP and GDP are as indicated. UI, unirradiated; 30 min after IR treatment as compared to the ML, mock lysate unirradiated control (GTP/GDP on Ras: unirradiated 100%; 30 min post-IR 256%). These data indicate that IR treatment increases the steady-state level of active IP: anti-Grb2 Ras as shown by the level of GTP bound to Ras and GTPase activity on Ras.

Blot: anti-SOS ¨ SOS Radiation stimulates association of Grb2 with Sos in vivo –+ Previous reports suggest that Grb2 serves as an adaptor that recruits Sos to the activated cell surface receptor

and Sos, in turn, catalyzes the exchange of GDP bound to Ras by GTP required for a positive regulation of Ras ¨ Blot: anti-Grb2 Grb2 activity (Lowenstein et al., 1992; Buday and Downward, 1993; Chardin et al., 1993; Gale et al., 1993). We asked whether IR exposure of MDA-MB 231 cells causes an –+ association between Grb2 and Sos. Representative data IR is shown in Figure 2. Whole cell lysates prepared at Figure 2 Radiation stimulates association of Grb2 with Sos in 15 min after IR were immunoprecipitated with anti- MDA-MB 231 cells. Cells were serum-starved, irradiated (15 Gy), Grb2 antibody. The Grb2 immunoprecipitates were and lysed at 15 min after IR. The normalized protein contents sequentially immunoblotted with anti-Sos antibody, (1 mg) were immunoprecipitated with anti-Grb2 antibody, and the Grb2 immune-complexes were immunoblotted with anti-Sos followed by anti-Grb2 antibody. Sos was found to co- MAb (top). The same blot was stripped and then reprobed with immunoprecipitate with Grb2 in irradiated cells. anti-Grb2 MAb (bottom). Data shown is representative of three Approximately twofold increase in Sos binding to independently performed experiments Radiation activates the Ras signaling pathway SSuyet al 55 antibody (238) was used because previously it has been to the membranes after IR exposure of MDA-MB 231 shown that it does not interfere with the direct cells, membrane fractions were collected at various interaction of Ras and Raf (Warne et al., 1993; times after irradiation and immunoprecipitated with Hallberg et al., 1994). The Ras immunoprecipitates anti Raf-1 antibody, followed by immunoblotting with were then immunoblotted with anti-Raf antibody. Raf- anti-Raf-1 antibody. Representative data is shown in 1 was found to co-immunoprecipitate with Ras in Figure 4. The immunoreactive bands were quanti®ed irradiated cells (Figure 3a, top). Approximately (ImageQuant software program, Molecular Dynamics). threefold increase in Raf binding to Ras was observed Approximately threefold increase in the amount of (Figure 3b). When the same blot was reprobed with membrane-bound Raf-1 protein was noted at 30 min anti-Ras antibody, no change in the levels of Ras after irradiation. These data suggest that Raf is an protein was noted after IR (Figure 3a, bottom). To e€ector of Ras in the IR-stimulated signal transduction rule out the possibility that Ras and Raf interaction pathway. may occur after lysis of the cells, increasing amounts of full length Raf-1-GST fusion protein (*95 kDa, Upstate Biotechnology Inc.) were added to irradiated cells at the time of lysis, and the Ras proteins were Post-IR Time immunoprecipitated and immunoblotted with anti-Raf- UI 5' 30' 90' 1 antibody. The GST-Raf failed to compete with endogenous Raf (*75 kDa) for binding to Ras, IP: anti-Raf-1 ¨ Raf-1 suggesting that the endogenous Raf ± Ras complex is Blot: anti-Raf-1 (~75 kDa) formed before cell lysis (data not shown). The Figure 4 Radiation stimulates membrane-recruitment of Raf-1 in association of Raf-1 with Ras was also noted when vivo. MDA-MB 231 cells were serum-starved, irradiated (15 Gy), the membrane fractions immunoprecipitated with anti- and lysed in homogenization bu€er at the indicated times after Ras antibody were immunoblotted with anti-Raf-1 irradiation as described in Materials and methods. Membrane antibody (Figure 3c, top). A 3 ± 4-fold increase in the fractions from irradiated and unirradiated cells were normalized amount of immunoreactive Raf-associated with Ras for protein contents (100 mg) and immunoprecipitated with agarose-conjugated anti-Raf-1 antibody, followed by immuno- was observed in the membranes after IR (Figure 3c, blotting with anti-Raf-1 MAb. Representative data from 2 ± 3 bottom). To further examine the translocation of Raf-1 experiments is shown. UI, unirradiated

a c IP: anti-Ras

IP: anti-Ras Blot: anti-Raf-1 ¨ Raf-1

(75 kDa) Blot: anti-Raf-1 ¨ ¨ Raf-1 IgG (~75 kDa) (H.C.) +– ML +–

IR ¨ IgG Blot: anti-Ras (L.C.)

¨ Ras ML +– (21 kDa) IR

b

Figure 3 Radiation stimulates association of Raf-1 with Ras in MDA-MB 231 cells. (a) Cells were serum-starved, irradiated (15 Gy), and lysed at 30 min after IR. The normalized protein contents (400 mg) were immunoprecipitated with anti-Ras antibody, and the Ras immune-complexes were immunoblotted with anti-Raf-1 MAb (top). The same blot was stripped and then reprobed with anti-Ras MAb (bottom). (b) The immunoreactive Raf-1 bands shown in (a) (top) were quanti®ed, and data shown are mean+s.d. from three independently performed experiments. (c) Cells were serum-starved, irradiated, and the membranes were isolated at the indicated times after 30 min. IR exposure as described in Materials and methods. Membrane fractions (100 mg) were immunoprecipitated with agarose-conjugated anti-Ras antibody, followed by immunoblotting with anti-Raf-1 MAb (top). The immunoreactive bands in the top panel were quanti®ed to determine the fold increase in the association of membrane-bound Raf-1 Radiation activates the Ras signaling pathway SSuyet al 56 Radiation stimulates tyrosine phosphorylation of Raf-1 increase, up to 30 min post-IR, was noted in the membrane-bound Raf-1 when anti-P-Y MAb immu- in MDA-MB 231 cells noprecipitates of the membrane fractions were Previous reports indicate that Ras binding is immunoblotted with anti-Raf-1 MAb (Figure 5b). insucient to activate Raf-1, and that active pool of Interestingly, the levels of Raf-1 detected in anti-P-Y Raf-1 is membrane-localized, tyrosine-phosphorylated MAb immunoprecipitates were signi®cantly higher as form of Raf-1 (Traverse et al., 1993; Jelinek et al., compared to the levels of tyrosine-phosphorylated 1996). To determine whether IR stimulates tyrosine Raf-1 identi®ed by anti-P-Y immunoblotting of the phosphorylation of Raf-1 in MDA-MB 231 cells, Raf-1 immunoprecipitates (Figure 5a and b). The WCE prepared after IR were immunoprecipitated with basis for this appears to be that a signi®cant portion anti-Raf-1 antibody, followed by immunoblotting with of the membrane-bound Raf-1 may be non-tyrosine anti-P-Y MAb (Figure 5a, top). The 75 kDa phospho- phosphorylated and associated with other as-yet protein band was con®rmed as Raf-1 (Raf-1P)by unknown tyrosine-phosphorylated proteins in irra- stripping the same blot and reprobing with anti-Raf-1 diated breast cancer cells. MAb (Figure 5a, middle). Several high molecular weight phospho-proteins co-immunoprecipitated with Radiation stimulates Raf-1 protein kinase activity in Raf-1 only after irradiation (Figure 5a: top, lane 3). vivo The signi®cance of these interactions remains to be de®ned. Data from four independent experiments To determine whether IR-stimulated activation of Ras indicate that a 3 ± 4-fold increase in Raf-1-phospho- and tyrosine phosphorylation of Raf-1 correlated with tyrosine occurred after IR in MDA-MB 231 cells. enzymatic activation of Raf-1 in MDA-MB 231 cells, Representative data is shown in Figure 5a, bottom. the catalytic activity of Raf-1 protein kinase was The levels of Raf-1 protein did not change after measured using its physiological substrate, mitogen- irradiation (Figure 5c). Furthermore, a steady activated protein kinase kinase (MAPK kinase,

ab IP: anti-Raf-1 IP: anti-P-Y Blot: anti-Raf-1

Post-IR Time Post-IR Time

kDa ML 180' 30' 15' UI 90' 30' 15' 10' 5' 1' UI 204 — Blot: anti-P-Y 144 — ¨ Raf-1 87 — (~75 kDa) ¨ Raf-1®P (~75 kDa) 54321 c IP: anti-Raf-1 Blot: anti-Raf-1 Post-IR Time Blot: anti-Raf-1 ¨ Raf-1 UI 5' 15' 30' 60'120'

54 321 ¨ Raf-1

–+

Figure 5 Radiation stimulates tyrosine phosphorylation of Raf-1 in MDA-MB 231 cells. (a) MDA-MB 231 cells were serum- starved, irradiated (15 Gy), and lysed at the indicated times after irradiation. WCE (*600 mg) were immunoprecipitated with agarose-conjugated anti-Raf-1 antibody, followed by immunoblotting with anti-P-Y MAb (top). The same blot was stripped and then reprobed with anti-Raf-1 MAb (middle). Left and right insets (bottom): Tyrosine-phosphorylated Raf-1 (75 kDa Raf-1P, shown by arrow heads) was detectable at 5 min (left) and at 30 min (right) after IR. The immunoreactive Raf-1P bands were quanti®ed and representative data from four independently performed experiments is shown (bottom). (b) In parallel experiments, membrane fractions (100 mg protein per sample) were immunoprecipitated with agarose-conjugated anti-P-Y MAb, followed by immunoblotting with anti-Raf-1 MAb. (c) The levels of Raf-1 protein do not change after IR treatment. WCE were prepared as in (a) and normalized protein contents were immunoprecipitated with anti-Raf-1 antibody, followed by immunoblotting with anti Raf- 1 MAb. UI, unirradiated: ML, mock lysate Radiation activates the Ras signaling pathway SSuyet al 57 MKK1; Dent et al., 1992; Howe et al., 1992; Kyriakis Radiation stimulates tyrosine phosphorylation and et al., 1992) (Figure 6). Densitometric analysis activity of MAPK in MDA-MB 231 cells indicated *twofold induction of MKK1 phosphoryla- tion in irradiated cells. A slight increase in the in vitro We next examined the e€ect of IR on MAPK activity in autokinase activity of Raf-1 was occasionally seen after MDA-MB 231 cells. WCE were prepared at various irradiation (data not shown). times after IR, followed by immunoprecipitation with anti-ERK-1 antibody. The ERK-1 (MAPK) immuno- precipitates were immunoblotted with anti-phospho- MAPK antibody (Figure 7a, top). Anti-phospho- MAPK antibody was used because it recognizes IP: anti-Raf-1 phosphorylated p42mapk and p44mapk (MAPKP). As kDa shown, a gradual increase in the amount of MAPKP was observed after IR exposure (Figure 7a, top). 86 — ¨ Raf-1 Reprobing of this blot with anti-ERK-1 antibody con®rmed that the bands at *44 kDa represent MAPK (Figure 7a, bottom). Densitometric scanning of the blot indicated a tenfold increase in phospho- P 50 — MAPK (MAPK ) at 2 h after IR (Figure 7b). To further ¨ MKK-1 show that tyrosine phosphorylation of MAPK caused its catalytic activation, MAPK activity was measured using myelin basic protein (MBP) as a substrate (Figure –+ 7c). The MBP phosphorylation in the presence of IR (15 Gy) MAPK immunoprecipitates increased by approxi- Figure 6 Radiation stimulates Raf-1 activity in MDA-MB 231 mately 12-fold after IR, consistent with the amount of cells. Cells were serum-starved, irradiated (15 Gy) and lysed after phospho-MAPK observed after IR (Figure 7b,c). A IR as explained in Materials and methods. WCE (1.2 mg per strikingly larger increase in MAPK activity after IR as sample) were immunoprecipitated with anti-Raf-1 antibody, and compared to the increase in Raf-1 activity implies that in vitro phosphotransferase activity of Raf-1 was assayed using a physiological substrate, MKK1. Radiolabeled reaction products IR may stimulate multiple signals upstream of MAPK, were separated by electrophoresis and autoradiographed some of which may be independent of Ras and Raf-1.

a b IP: anti-ERK-1 Post-IR Time

120' 60' 15' 10' UI

®P P ¨ MAPK Blot: anti-MAPK® (~44 kDa) 5432 1

Blot: anti-ERK-1 ¨ ERK-1 (~44 kDa)

5432 1

c

Figure 7 Radiation stimulates tyrosine phosphorylation and activity of MAPK in MDA-MB 231 cells. (a) Cells were serum- starved, irradiated (15 Gy), and lysed at the indicated times after IR exposure. WCE were normalized for protein content (600 mg) and immunoprecipitated with agarose-conjugated anti-ERK-1 antibody which recognizes both p42mapk (ERK-2) and p44mapk (ERK- 1). The MAPK immune-complexes were immunoblotted with anti-phospho-MAPK MAb (top). The same blot was stripped and reprobed with anti-ERK-1 MAb (bottom). (b) Quanti®cation of the immunoreactive phospho-MAPK bands in (a) (top). (c)IR stimulates MBP kinase activity of MAP kinase. MDA-MB 231 cells were serum-starved, irradiated (15 Gy), and lysed at the indicated times after IR exposure. WCE were normalized for protein content (600 mg) and immunoprecipitated with agarose- conjugated anti-ERK-2 antibody which recognizes both p42mapk and p44mapk. The MAPK immune-complexes were incubated with 5 nmol of [g-32P]ATP and 20 mg of MBP in 40 ml of the kinase reaction bu€er, and MBP phosphorylation was measured in a ®lter binding assay as described in Materials and methods. Representative data shown are mean+s.d. from triplicate samples. UI, unirradiated Radiation activates the Ras signaling pathway SSuyet al 58

Figure 8 A model of ionizing radiation-stimulated Ras signaling pathway. Irradiation of cells causes increases in Ras.GTP via Grb2-mediated activation of Ras nucleotide exchanger Sos. Activation of Ras leads to Raf-1 activation by membrane-recruitment and tyrosine phosphorylation, as well as to downstream phosphorylation and activation of MEK (MKK) and MAPK

Discussion compared to vector control cells (Gibbs et al., 1990). Whether an increase in GAP activity after IR decreases In this report, we demonstrate for the ®rst time that IR the steady-state level of Ras.GTP remains to be seen. exposure of cells stimulates a Ras signaling pathway Our ®ndings provide further impetus in the search for leading to the activation of MAPK (Figure 8). the nucleotide exchange regulatory mechanisms in Association of Grb2 with Sos, increase in Ras.GTP, breast cancer cells. association of Ras with Raf-1 and tyrosine phosphor- Raf-1 phosphotyrosine generated after IR as shown ylation of Raf-1 after irradiation were all observed in Figure 5, may be due to membrane-localized events with an identical post-IR timing i.e. *30 min (Figures such as an IR-stimulated PTK(s). Mutations in Raf-1 1 ± 6). Surprisingly, a majority of the IR-stimulated have been shown to abrogate its association with Ras, biochemical responses observed were modest (2 ± 4-fold and mutations of tyrosines at positions 340 and 341 to above the unirradiated controls). This may be, in part, phenylalanine create an inactive Raf-1 protein (Fabian due to the fact that radiation lacks target speci®city et al., 1993, 1994). It is likely that these sites on Raf-1 and may initiate multiple signaling pathways, not all of may be critical for IR stimulation of the Ras ± Raf-1 which are convergent. interaction, and Raf-1 tyrosine phosphorylation. Activation of the Ras.GTP complex could involve Previous reports suggest that the protein kinase Ca either stimulation of guanine nucleotide exchange (PKC)-induced phosphorylation of Raf-1 increases factor activity or inhibition of GTPase activating autophosphorylation activity of Raf-1 (Kolch et al., protein (p120GAP) activity (Trahey and McCormick, 1993; Sozeri et al., 1992). Other studies in vitro 1987). The e€ects of IR on the association and demonstrate that although PKC phosphorylation of dissociation of Sos-Grb2, and GAP activity are not Raf-1 stimulates its autokinase activity, it does not known. Di€erent agents (insulin, serum, PDGF) that stimulate its ability to phosphorylate MKK (Macdo- activate Ras-MAPK pathway induce phosphorylation nald et al., 1993). The present data do not rule out the of Sos resulting in dissociation of the Grb2-Sos possibility of Raf-1 phosphorylation and activation as complex (Waters et al., 1995). In breast carcinoma a result of other IR-stimulated protein kinase(s) or cells, it is likely that the EGF or Neu receptors are -inactivated protein phosphatase(s). involved in signaling via Ras. However, although EGF To corroborate previous ®ndings from our labora- induces MAPK activation and Sos phosphorylation; in tory and others that IR stimulates protein tyrosine most cases, EGF is unable to induce uncoupling of phosphorylation of several unidenti®ed proteins, whole Grb2-Sos (Holt et al., 1996). In NIH3T3 cells cell lysates were prepared at various times after IR and overexpressing GAP, GAP decreased the PDGF- immunoprecipitated with anti-P-Y antibody. This was stimulated steady-state level of the Ras.GTP complex followed by immunoblotting of the anti-P-Y immuno- Radiation activates the Ras signaling pathway SSuyet al 59 precipitates with anti-P-Y antibody. An increase in the Minimum Essential Medium (IMEM) (Cellgro) containing levels of tyrosine phosphorylation of several proteins 10% bovine calf serum and 2 mML-glutamine in a was detected after IR treatment of breast cancer cells humidi®ed atmosphere of 5% CO2 : 95% air at 378C. Cells (data not shown). Furthermore, several high molecular were trypsinized and plated onto a 150 mm tissue culture weight phosphoproteins were found to co-immunopre- dish (one dish per ¯ask) overnight in 10% serum containing medium, followed by washing three times with cipitate with Raf-1 after exposure of these cells to phosphate bu€er saline (PBS) and maintaining cultures in radiation. Interestingly, these proteins also were serum-free medium for 18 h prior to irradiation. Irradia- tyrosine-phosphorylated in a radiation-dependent tions (15 Gy, 114 cGy/min) were performed in a JL manner (Figure 5a, top). We do not as-yet know the Shepherd Mark I Radiator using 137Cesium as the identity of these phosphoproteins, and whether they radiation source. After irradiation, cells were washed bind to tyrosine-phosphorylated Raf-1, or to the highly three times with ice-cold PBS containing 0.5 mM sodium abundant non-tyrosine-phosphorylated Raf-1 which orthovanadate [Na3VO4] and lysed in either lysis bu€er for also is immunoprecipitated. Such information may the isolation of WCE or in homogenization bu€er for the facilitate understanding of additional components of membrane isolation. Cells from six 150 mm dishes were IR signaling cascade. pooled for each time point following radiation treatment. WCE lysis bu€er consisted of 50 mM HEPES, pH 7.5, 1% In the present study, a *12-fold induction of the Nonidet P-40 [NP-40], 10% glycerol, 4 mg of leupeptin per amount of phospho-MAPK, and a similar level of the ml, 4 mg of aprotinin per ml, 4 mg of pepstatin A per ml, induction of the MBP-kinase activity of MAPK was 1mM Na3VO4,1mM phenylmethylsulfonyl ¯uoride noted at 2 h after irradiation of MDA-MB 231 cells [PMSF], 25 mM sodium ¯uoride [NaF], and 0.5 mM (Figure 7). This is in contrast to the kinetics of the EDTA. Homogenization bu€er consisted of 10 mM Tris- association between Raf-1 and Ras, and activation of HCl [pH 7.4], 0.5 mM EDTA, 0.3 M sucrose, 4 mgof Raf-1, where near basal levels were restored at 60 min leupeptin per ml, 4 mg of pepstatin A per ml, 4 mgof after irradiation (data not shown). One possible aprotinin per ml, 100 mg of PMSF per ml, 200 mM Na3VO4, explanation for the relatively sustained activation of and 25 mM NaF. MAPK is that IR may stimulate both Ras/Raf-1- dependent and independent pathways upstream of Membrane isolation MAPK. Depending on the cellular context and extracellular Cell membrane isolation was carried out according to the published procedure (Traverse et al., 1993) with the signal, the Ras/Raf-1 protein kinase cascade has been following modi®cations. Brie¯y, irradiated cells and shown to regulate cell proliferation, di€erentiation, and unirradiated control cells (UI) were homogenized in 5 ml development (Egan and Weinberg, 1993; Marshall, 1995). of homogenization bu€er with a tight ®tting Wheaton- The overexpression and activation of Ras and Raf-1 have Dounce hand homogenizer and centrifuged at 48Cfor been associated with increased survival of mammalian 10 min at 3000 g to remove nuclear debris followed by cells exposed to IR (Kasid et al., 1989, 1993; Pirollo et al., another 15 min centrifugation at 5000 g to remove 1989; McKenna et al., 1990; Bernhard et al., 1996; mitochondria. The supernatant was then centrifuged at Soldatenkov et al., 1997), underscoring the importance 48C for 60 min at 100 000 g to collect the membrane pellet. ofsignalingthroughRas ± Rafinthepossibleregulationof The membrane-enriched fractions were washed once with IR response. The primary target(s) of Raf-1 in response to homogenization bu€er without sucrose followed by centrifugation at 15 000 g for 15 min. Membrane-enriched radiation is not known. Future investigations of the fractions were then resuspended in 100 ml homogenization regulators and e€ectors of Raf-1 should provide insight bu€er without sucrose and stored at 7708C until use. into the diverse mechanisms of IR-induced cell transfor- mation and cell death. Immunoprecipitation and immunoblotting Whole cell lysates or membrane fractions were immuno- Materials and methods precipitated with the appropriate agarose-conjugated antibodies (1 mg per ml of lysis bu€er) overnight at 48C with constant agitation. The beads containing the immune- Antibodies complexes were collected by centrifugation at 15 000 g for The following antibodies were used in this study: anti- 5 min followed by three washes with the lysis bu€er. The phosphotyrosine (P-Y) monoclonal antibody (MAb) and beads were resuspended in 26electrophoresis sample bu€er agarose-conjugated anti-P-Y MAb 4G10 (UBI, Lake and boiled for 5 min. Anti-Raf-1 immunoprecipitates were Placid, NY); agarose-conjugated anti-Grb2 antibody resolved by 7.5% sodium dodecyl sulfate-polyacrylamide (Santa Cruz Biotechnology); anti-Grb2 mouse MAb gel electrophoresis [SDS ± PAGE] for immunoblotting with (Transduction Laboratories); anti-Sos mouse MAb anti-Raf-1 MAb, anti-Grb2 immunoprecipitates were (Transduction Laboratories); agarose-conjugated anti-v- resolved on a step gradient of 5% SDS ± PAGE (top half) H-Ras MAb (238), agarose-conjugated anti-H-Ras MAb and 10.5% SDS ± PAGE (bottom half) for sequential (259), anti-Raf-1 (C-12), anti-ERK-1 (C-16) and anti-ERK- immunoblotting with anti-Sos antibody followed by anti- 2 antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, Grb2 antibody, anti-Ras immunoprecipitates were electro- CA); anti-pan-Ras MAb (Ab-4) (Oncogene Science, phoresed on a step gradient of 10% SDS ± PAGE (top half) Uniondale, NY); anti-Raf-1 MAb (c-Raf-1) and anti- and 12.5% SDS ± PAGE (bottom half) for immunoblotting ERK-1 MAb (MK12) (Transduction Laboratories, Lex- with anti-Ras and/or anti-Raf-1 MAbs, and anti-ERK-1 ington, Kentucky); and anti-phospho MAPK Ab (New immunoprecipitates were resolved by 12.5% SDS ± PAGE England Biolabs, Beverly, MA). for immunoblotting with anti-ERK-1 MAb. Following electrophoresis, samples were transferred to an Immobi- lon-P membrane and immunoblotted with the desired Cell culture and radiation treatment primary antibody at 1 : 2000 dilution, followed by MDA-MB 231 human breast cancer cells were grown to 1 : 10 000 dilution of an appropriate secondary antibody near con¯uence in 75 cm2 tissue culture ¯asks in improved coupled to horseradish peroxidase (goat anti-mouse IgG/ Radiation activates the Ras signaling pathway SSuyet al 60 anti-rat IgG/anti-rabbit IgG; Jackson Immuno Research ferase activity was assayed in vitro using mitogen-activated Laboratories Inc, West Grove, PA). Immunoreactive protein kinase kinase (MKK1) as substrate in kinase bu€er proteins were detected by enhanced chemiluminescence containing 30 mM HEPES [pH 7.4], 1 mM manganese (ECL) (Amersham). The immunoreactive bands of interest chloride, 15 mM magnesium chloride, 1 mM DTT, 0.1 mM were quanti®ed by ImageQuant software version 3.3 ATP, and 20 mCi [g-32P]ATP (6000 Ci/mmol) as described (Molecular Dynamics Personal Densitometer). For reprob- before (Kasid et al., 1996). Radiolabelled reaction products ing of the blots with a di€erent primary antibody, blots were separated by 10% SDS ± PAGE and autoradio- were stripped as described in the ECL kit protocol (NEN). graphed. MKK1 band was quanti®ed (ImageQuant soft- Brie¯y, after the ®rst ECL detection step, membrane was ware program). washed four times, 5 min for each wash, in 0.25% Tween- 20 in PBS (PBS-T), followed by incubation of the MAP kinase activity assay membrane for 30 min at 508C in stripping bu€er (62.5 mM Tris-HCl [pH 6.8], 2% SDS, and 100 mM 2b- Whole cell lysates (1 mg protein per time point) were mercaptoethanol). The stripped membrane was washed six immunoprecipitated with agarose-conjugated anti-ERK-2 times, 5 min each wash, in PBS-T. To ensure that the antibody (1 mg) in 1 ml of lysis bu€er overnight at 48C antibodies were completely stripped o€, the stripped blot with constant agitation. The ERK-2 immune-complexes was redeveloped by the ECL method, followed by exposing were washed three times with lysis bu€er and assayed for of the blot to X-ray ®lm for 30 min. The blot was then MAP kinase activity according to the MAPK assay kit washed four times, 5 min each wash, in PBS-T. protocol (UBI). Brie¯y, the ERK-2 immunoprecipitates were washed once with assay dilution bu€er (ADB: 20 mM MOPS [pH 7.2], 25 mM b-glycerol phosphate, 5 mM Assessment of Ras activity EGTA, 1 mM Na3VO4,and1mM DTT). The immune- GTP binding and GTPase activity of Ras in irradiated and complexes were resuspended in 10 mlofADB.The unirradiated cells were determined as described previously reactions were initiated by addition of 10 mlofthe (Downward, 1995). Brie¯y, WCE (1 mg) were immunopre- substrate cocktail (2 mg myelin basic protein (MBP) per cipitated overnight with 1 mg of agarose-conjugated anti- ml of ADB), 10 ml of the inhibitor cocktail (20 mM PKC H-Ras (259) monoclonal antibody. Immune-complexes inhibitor peptide, 2 mM protein A inhibitor peptide (PKI), were washed three times with lysis bu€er and once with and 20 mM compound, R24571), and 10 mlofthe 32 exchange bu€er (50 mM HEPES [pH 7.5], 1 mM magne- magnesium-ATP cocktail (1 mCi [g- P]ATP (3000 Ci/ sium chloride, 1 mM dithiothretiol (DTT), 100 mM mmol) in 75 mM magnesium chloride and 500 mM cold potassium chloride, 0.1 mg of bovine serum albumin per ATP). The reaction mixtures were then incubated for ml). The hydrolysis of bound GTP in Ras immunopreci- 15 min at 308C with constant agitation. The reaction was pitates was determined in an exchange reaction by the stopped by blotting 10 ml of the reaction mixture, in addition of 10 ml of exchange bu€er containing 1 mCi of triplicate, onto P81 phosphocellulose paper. The ®lter [a-32P]GTP (3000 Ci/mmol) and incubation at 378Cfor papers were air dried and washed three times in a 50 ml 5 min. This was followed by addition of 2 mlof60mM conical tube containing 40 ml of 0.75% phosphoric acid magnesium chloride and 1 mM cold GDP and continued with gentle shaking on a rotator followed by an acetone incubation for 30 min at 208C with constant agitation. The wash. The ®lters were air dried and transferred to a radiolabeled GTP and GDP bound were eluted with scintillation vial containing 5 ml of scintillation ¯uid. The 0.5 mM cold GTP and 0.5 mM cold GDP for 20 min at radioactivity bound to the ®lters was determined using a 688C. Eluted samples were spotted on polyethyleneimine Beckman scintillation counter. (PEI) cellulose plates (Sigma), and the released nucleotides were resolved by thin layer chromatography in 0.75 M

dibasic potassium phosphate [KH2PO4] [pH 3.4] and visualized by autoradiography. GTP and GDP spots were Acknowledgements quanti®ed (ImageQuant software version 3.3, Molecular We thank T Sturgill and D Morrison for helpful discus- Dynamics). sions, D Massaro for access to the ImageQuant software program for densitometry. Puri®ed MKK1 was kindly Raf-1 kinase activity assay obtained from T Sturgill's laboratory. MDA-MB 231 cells were obtained from the Tissue Culture Core Facility of the Whole cell extracts (1 mg protein per ml of lysis bu€er) Lombardi Cancer Center (NIH grant P30 CA51008). This were immunoprecipitated with agarose-conjugated anti- work was supported by the National Institutes of Health Raf-1 antibody overnight at 48C and Raf-1 phosphotrans- Grants CA58984 and CA68322 (to UK).

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