Radiation-induced Epidermal Growth Factor Receptor Nuclear Import Is Linked to Activation of DNA-dependent Protein Kinase

Abstract

Ionizing radiation, but not stimulation with epidermal growth factor (EGF), triggers EGF receptor (EGFR) import into the nucleus in a probably karyopherin ␣-linked manner. An increase in nuclear EGFR is also observed after treatment with H 2 O 2 , heat, or cisplatin. During this process, the proteins Ku70/80 and the protein phosphatase 1 are transported into the nucleus. As a consequence, an increase in the nuclear kinase activity of DNA-dependent kinase (DNA-PK) and increased formation of the DNA end-binding protein complexes containing DNA-PK, essential for repair of DNA-strand breaks, occurred. Blockade of EGFR import by the anti-EGFR monoclonal antibody C225 abolished EGFR import into the nucleus and radiation-induced activation of DNA-PK, inhibited DNA repair, and increased radiosensitivity of treated cells. Our data implicate a novel function of the EGFR during DNA repair processes.The epidermal growth factor receptor (EGFR) 1 is essential for mediation of both proliferative and survival signals to cells (1). At least five mitogenic growth factors bind to and activate EGFR. In addition to EGF these factors include transforming growth factor-␣, amphiregulin, heparin binding EGF (2), and epiregulin (3). Moreover, in recent years it became apparent that in addition to ligand binding-induced activation of the EGFR, ligand-independent receptor activating processes also exist (4). Activation of the EGFR signaling pathway by ligands has been linked with increased cell proliferation, angiogenesis, and metastasis and decreased apoptosis (5). As a consequence it is assumed that increased EGFR signaling plays an important role in tumorigenesis (6, 7). The mechanisms of ligand-independent EGFR activation and also the importance for the cell fate are not understood so far. But it is noteworthy that the ligand-independent activation always is linked to exposure to genotoxic stress (4,8,9). However, the differences of the observed cell response after ligand-induced EGFR activation, e.g. cell proliferation, and ligandindependent activation, e.g. cell cycle arrest, argue rather for a link to DNA repair processes for ligand-independent activation. This idea was strengthened by the observation that many tumor cells show an increased radiosensitivity after inhibition of EGFR signaling (10). The most detrimental DNA damages after treatment with ionizing radiation are double-strand strands (11), which are preferentially repaired in mammalians by nonhomologous end-joining (12). Looking for a connection between radiation-induced EGFR activation and DNA repair, we found a report stating that there is a physical interaction of EGFR and DNAdependent kinase (DNA-PK) (13) after cell treatment with the EGFR blocking antibody C225. As a consequence, the nuclear DNA-PK protein and activity was reduced, providing an explanation for the radiosensitizing effect of the EGFR blockage. However, this paper did not answer the question of the functional role of the physical interaction between EGFR and DNA-PK. Therefore, we addressed herein the...

Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival

“…In addition, EGFR has been shown to localize in the inner nuclear membrane (Cao et al, 1995;Klein et al, 2004). Nuclear counterpart of EGFR appears to be the full-length receptor and likely, in the phosphorylated form, as shown by a number of studies (Cao et al, 1995;Lin et al, 2001;Cordero et al, 2002;Dittmann et al, 2005a;Lo et al, 2005a, c). Consistently, EGF and pro-TGF-a were found to translocate into the nucleus of proliferating hepatocytes (Raper et al, 1987;Schausberger et al, 2003).…”

“…Consistently, EGF and pro-TGF-a were found to translocate into the nucleus of proliferating hepatocytes (Raper et al, 1987;Schausberger et al, 2003). In addition to ligand stimulation, nuclear translocalization of EGFR can be initiated by irradiation, heat shock, H 2 O 2 and cisplatin (Cao et al, 1995;Lin et al, 2001;Dittmann et al, 2005a). Conversely, EGF-and irradiation-induced EGFR nuclear transport can be blocked by 1,25-dihydroxyvitamin D (Cordero et al, 2002) and anti-EGFR antibody, C225/Cetuximab (Dittmann et al, 2005b), respectively.…”

“…Several lines of evidences suggest that many nuclear RTKs can be originated from the cell-surface counterparts and exist as uncleaved full-length receptors, including EGFR, HER-2, HER-3, and FGFR1 (Maher, 1996;Lin et al, 2001;Offterdinger et al, 2002;Wang et al, 2004;Dittmann et al, 2005a;Giri et al, 2005; Following nuclear translocalization, nuclear EGFR interacts with DNA-binding transcription factors, E2F1 and STAT3, and activates expression of B-Myb and iNOS, respectively. Nuclear EGFR also upregulates cyclin D1 gene expression.…”

“…In the case of HER-2 which lacks the domain/ability for ligand-binding, its kinase activity is required for nuclear entry (Wang et al, 2004). Nuclear import of EGFR and type I TGF-b receptor can occur in a ligand-independent manner (Zwaagstra et al, 2000;Dittmann et al, 2005a). In contrast, heregulin b1 activates nuclear export of HER-3 in mammary epithelial cells, which contain high levels of nuclear HER-3 under normal growth conditions (Offterdinger et al, 2002).…”

“…Nevertheless, it is becoming clear that several cellsurface receptors are capable of interacting with nuclear transport receptors, importins a/b and exportins, and thus enter and exit the cell nucleus, respectively (Reilly and Maher, 2001;Giri et al, 2005). Association with importin b1 is important for nuclear import of HER-2 and FGFR (Reilly and Maher, 2001;Giri et al, 2005) whereas EGFR has been shown to interacts with both importin b1 and importin a (Dittmann et al, 2005a;. Blockage of RanGDP and importin b1 by dominant-negative mutant and siRNA, respectively, also inhibits nuclear transport of HER-2, suggesting that NPC is involved in the process (Giri et al, 2005).…”