Inhibition of protein synthesis per se does not potentiate the stress-activated protein kinases (SAPKs; also known as cJun NH 2 -terminal kinases [JNKs]). The protein synthesis inhibitor anisomycin, however, is a potent activator of SAPKs/JNKs. The mechanism of this activation is unknown. We provide evidence that in order to activate SAPK/JNK1, anisomycin requires ribosomes that are translationally active at the time of contact with the drug, suggesting a ribosomal origin of the anisomycin-induced signaling to SAPK/JNK1. In support of this notion, we have found that aminohexose pyrimidine nucleoside antibiotics, which bind to the same region in the 28S rRNA that is the target site for anisomycin, are also potent activators of SAPK/JNK1. Binding of an antibiotic to the 28S rRNA interferes with the functioning of the molecule by altering the structural interactions of critical regions. We hypothesized, therefore, that such alterations in the 28S rRNA may act as recognition signals to activate SAPK/JNK1. To test this hypothesis, we made use of two ribotoxic enzymes, ricin A chain and ␣-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA. Consistent with our hypothesis, ricin A chain and ␣-sarcin were strong agonists of SAPK/JNK1 and of its activator SEK1/MKK4 and induced the expression of the immediate-early genes c-fos and c-jun. As in the case of anisomycin, ribosomes that were active at the time of exposure to ricin A chain or ␣-sarcin were able to initiate signal transduction from the damaged 28S rRNA to SAPK/JNK1 while inactive ribosomes were not.The activity of the stress-activated protein kinases (SAPKs; also known as cJun NH 2 -terminal kinases [JNKs]) is stimulated in response to certain kinds of cellular stress, including exposure of cells to short-wavelength UV radiation (11, 19), alkylating DNA-damaging agents (27), the tumor promoters As 3ϩ (7) and palytoxin (23), hyperosmotic shock (16), proinflammatory cytokines (24), or withdrawal of a trophic factor (54). SAPKs/JNKs are members of the mitogen-activated protein kinase (MAPK) family of proline-directed serine/threonine protein kinases, which also includes the extracellular signalregulated kinases (ERKs) and the p38/RK/HOG1 kinase (for a review, see reference 51). Upon activation, SAPKs/ JNKs phosphorylate and activate transcription factors such as cJun (11), ATF-2 (17, 49), and Elk-1 (6, 52, 56), leading ultimately to the transcriptional activation of the immediate-early genes c-fos and c-jun (49, 56). The signal transduction cascades that lead to activation of SAPKs/JNKs and to subsequent gene induction are thought to be associated with stress responses that promote either cell recovery and survival after cellular damage (13,18,41) or, in some instances, apoptotic death (8, 54). The activity of SAPKs/JNKs is regulated through their phosphorylation on both threonine and tyrosine residues in the motif T‫ء‬PY‫ء‬ by the dual-specificity protein kinase SEK1/MKK4 (12,26,40). The protein kinase MEKK1 (25), in turn, activates SEK1/MK...

The ribotoxic stress response, which is conserved between prokaryotes and eukaryotes, is a cellular reaction to cytotoxic interference with the function of the 3-end of the large (23 S/28 S) ribosomal RNA. The 3-end of the large rRNA is directly involved in the three sequential steps of translational elongation: the aminoacyl-tRNA binding, the peptidyl transfer, and the ribosomal translocation. In mammalian cells, the ribotoxic stress response involves activation of the stress-activated protein kinase/c-Jun NH 2 -terminal kinase and the p38 mitogen-activated protein kinase and transcriptional induction of immediate early genes such as c-fos and c-jun. Active ribosomes are essential mediators of the ribotoxic stress response. We demonstrate here that the transcriptional response of mammalian cells to ultraviolet radiation (UV response) displays the characteristics of a ribotoxic stress response, inasmuch as (i) the activation of stress kinases and gene expression in response to UV requires the presence of active ribosomes at the moment of irradiation; (ii) UV irradiation inhibits protein synthesis; and (iii) irradiation of cells with UV causes specific damage to the 3-end of the 28 S rRNA. In contrast, the activation of the stress kinases by hyperosmolarity, by the DNA-cross-linking agent diepoxybutane, or by growth factors and cytokines does not depend on the presence of active ribosomes. Our results identify UV as a potential ribotoxic stressor and support the notion that some of the cellular signaling cascades in response to UV might be generated in the ribosome, possibly triggered by damage to rRNA.

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH 2 -terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L-and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections. Double-stranded RNA (dsRNA) produced during viral infections triggers stress response pathways that lead to elimination of infected cells by apoptosis. Two complementary but independent cellular dsRNA-detecting systems have been implicated in the translational inhibition in response to viral infection: the 2-5A system and the dsRNA-activated protein kinase (PKR) (for a recent review, see reference 55). The 2-5A system is composed of a family of dsRNA-dependent enzymes known as 2Ј-5Ј oligoadenylate synthetases (OAS) (5) and the dormant cytosolic RNase L (64) (for recent reviews on the 2-5A system and RNase L, see references 45 and 52, respectively). Upon dsRNA binding, OAS produce unusual second messengers, short 2Ј-5Ј-linked oligoadenylates (2-5A) (32), which, in turn, specifically bind to and activate RNase L (64). Activated RNase L cleaves diverse RNA substrates...

The down-regulation and internalization of the epidermal growth factor (EGF) receptors induced by two separate anti-EGF monoclonal antibodies (mAbs), IgG1 mAb-225 and -455, and by, EGF was examined. mAb-225 competitively inhibits EGF binding and it is internalized to an extent comparable to EGF. The antibody down-regulates surface EGF receptors in a dose-dependent manner. In contrast, mAb-455 does not competitively inhibit the binding of EGF or mAb-225, but it specifically immunoprecipitates the EGF receptor. mAb-455 also down-regulates the EGF receptor. Unlike EGF, which elicits phosphorylation of the receptor at tyrosine, threonine, and serine residues, neither of these antibodies elicits phosphorylation of the EGF receptor in intact A431 cells or in KB cells. Our studies suggest that EGFstimulated phosphorylation of the receptor is not required for the internalization of the ligand-receptor complex.Epidermal growth factor (EGF) is a potent mitogen for certain cultured cells and has been used extensively as a model for studying growth control (1, 2). Specific saturable receptors for EGF are present on a wide variety of tissues, including corneal cells, fibroblasts, lens glial cells, epidermoid carcinomas, granulosa cells, vascular endothelial cells, and choriocarcinomas (3,4). EGF is mitogenic for a wide variety of tissue culture cells. After EGF receptor binding, the EGF receptor kinase autophosphorylates itself at at least three sites near the carboxyl terminus (5,6). Phosphorylation of the receptor and other cellular substrates occurs at tyrosine residues (7,8). The occupied receptors cluster in clathrin-coated pits and are internalized into endocytic vesicles; ultimately, EGF is degraded in lysosomes (1, 3). The relationship between phosphorylation of the receptor and internalization has not been established, although both occur within a few minutes of EGF binding.Recent studies have shown that several human tumors express an increased number of EGF receptors (9-11). In some cases, as in A431 and human lung tumors, the increased receptor number is due to amplification of the EGF receptor gene (11-13).A recent study from Pastan's laboratory (14) indicates that EGF induces internalization of the EGF receptor into receptosomes, followed by the loss of immunoreactive EGF receptor from the lysosome. Although the initial hormonereceptor interactions at the plasma membrane are required to obtain a biological response, the relevance of internalization of the hormone-receptor complex into the cell and the fate of the internalized receptor are not yet established.The A431 epidermoid carcinoma cell line is unusual in that it displays an extremely high number of EGF receptors (15), and yet its growth is inhibited by concentrations of EGF that are mitogenic to other cell lines (16,17 Cells were placed on ice and washed three times with 0.5 ml of chilled DMEM/F-12 containing 0.2% bovine serum albumin and 0.02% NaN3 (buffer A) and incubated with 0.5 ml of 50 nM 1251I-labeled EGF at 4°C for 1.5 hr. Cell-associated...

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