Oscillations in the activation of multiple signaling pathways have never been shown before. Our results presented in the previous accompanying paper showed that TNF induces highly dynamic oscillations in mRNA production in similar to 13% of the mouse genome. Here, we further analyze the TNF time-series microarray data and find that multiple signaling components downstream of the TNF receptor undergo oscillations. Prior studies implicate I kappa B alpha and A-20 as the only oscillatory components in the TNF signaling cascade. We find however, that other components, such as TRAF1, displayed oscillations. This suggested the possibility that all signaling output from the TNF receptor may be oscillatory in nature. Indeed, we show that TNF triggers oscillations in the phosphorylation of three MAP kinases, as well as p65. Because Baltimore and colleagues had proposed that NF-kappa B drives the oscillatory nature of the I kappa B alpha/NF-kappa B feedback loop, we studied the effects of an NF-kappa B super-repressor on oscillations in MAPK phosphorylation; we find that the super-repressor altered the amplitude and frequency of MAP kinase phosphorylation, but failed to abolish oscillations. These results attest to a role for NF-kappa B as a modulator, but not the sole determinant of cyclical activation of signal transduction pathways. These results, together with those of the two accompanying papers, constitute a new paradigm through which cells orchestrate signaling molecules to produce highly dynamic physiological processes such as gene transcription and protein secretion. In view of the discovery that multiple phosphorylation pathways display dynamic oscillations, time-resolved, instead of static, measurements of kinase phosphorylation should become the experimental norm. (C) 2008 Elsevier Inc. All rights reserved.