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...

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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.

The importance of the dopaminergic system in brain function has been emphasized by its association with neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. On the basis of their biochemical and pharmacological characteristics, dopamine receptors are classified into D1 and D2 subtypes. As the most abundant dopamine receptor in the central nervous system, D1 receptors seem to mediate some behavioural responses, modulate activity of D2 dopamine receptors, and regulate neuron growth and differentiation. The D dopamine receptor has been cloned by low-stringency screening. We report here the cloning of human and rat D1 dopamine receptors by applying an approach based on the polymerase chain reaction. The cloned human D1 dopamine receptor has been characterized on the basis of four criteria: the deduced amino-acid sequence, which reveals that it is a G protein-coupled receptor; the tissue distribution of its messenger RNA, which is compatible with that of the D1 dopamine receptor; its pharmacological profile when transfected into COS-7 cells; and its ability to stimulate the accumulation of cyclic AMP in human 293 cells.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.