Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH 2 -terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0 -10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90 -240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor ␣ receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor ␣ (TGF␣) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGF␣ cleavage 120 -180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGF␣. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGF␣. Neutralization of TGF␣ function by an anti-TGF␣ antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGF␣-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.© 1999 by The American Society for Cell Biology 2493 INTRODUCTIONIonizing radiation has been shown to activate multiple signaling pathways within cells in vitro, which can lead to either increased cell death or increased proliferation depending on the cell type, the radiation dose, and the culture conditions (Xia et al., 1995;Rosette and Karin, 1996;Santana et al., 1996;Chmura et al., 1997;Schmidt-Ullrich et al., 1997;Carter et al., 1998;Haimovitz-Friedman, 1998;Kavanagh et al., 1998). Recently, a novel cellular target for ionizing radiation has been shown to be the ...

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Activation of the epidermal growth receptor (ErbB1) occurs within minutes of a radiation exposure. Immediate downstream consequences of this activation are currently indistinguishable from those obtained with growth factors (GF), e.g. stimulation of the pro-proliferative mitogenactivated protein kinase (MAPK). To identify potential di erences, the e ects of GFs and radiation on other members of the ErbB family have been compared in mammary carcinoma cell lines di ering in their ErbB expression pro®les. Treatment of cells with EGF (ErbB1-speci®c) or heregulin (ErbB4-speci®c) resulted in a hierarchic transactivations of ErbB2 and ErbB3 dependent on GF binding speci®city. In contrast, radiation indiscriminately activated all ErbB species with the activation pro®le re¯ecting that cell's ErbB expression pro®le. Downstream consequences of these ErbB interactions were examined with MAPK after speci®cally inhibiting ErbB1 (or 4) with tyrphostin AG1478 or ErbB2 with tyrphostin AG825. MAPK activation by GFs or radiation was completely inhibited by AG1478 indicating total dependance on ErbB1 (or 4) depending on which ErbB is expressed. Inhibiting ErbB2 caused an enhanced MAPK response simulating an ampli®ed ErbB1 (or 4) response. Thus ErbB2 is a modulator of ErbB1 (or 4) function leading to di erent MAPK response pro®les to GF or radiation exposure. Oncogene (2001) 20, 1388 ± 1397.

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras-and v-Src-transformed cells (transformed membranes). Wild-type (His) 6 -and FLAG-Raf-1 were activated in a Ras-and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and Src Y527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.The proto-oncogene product Raf-1 is a member of a family of serine/threonine protein kinases (Raf-1, B-Raf, and A-Raf) that function in protein kinase cascades important for mitogenic signaling (31). Raf-1 phosphorylates and activates mitogen-activated protein (MAP) kinase kinase (MKK, also known as MEK), the specific activator of MAP kinase (23). MAP kinase in turn phosphorylates several regulatory proteins (4, 21) in the cytoplasm (e.g., PHAS1, p90 rsk , and cPLA 2 ) and nucleus (e.g., p62TCF and c-Myc) to alter the program of transcription and translation required for mitogenesis. Raf-1 exhibits restricted substrate specificity in vitro (12). Although other in vivo substrates for Raf-1 than MKK may exist, none have been definitively established.The mechanism of Raf-1 activation is incompletely understood. Activation of Raf-1 in vivo occurs at the plasma membrane (20, 28) and is dependent upon association with Ras-GTP (24). Association of purified Raf-1 and Ras proteins in the presence of ATP in vitro is not sufficient to cause activat...