Analyzing the signaling capabilities of soluble and membrane TWEAK.

: TWEAK, like many other ligands of the TNF family, occurs naturally in two forms, as a type II transmembrane protein and as soluble ligand released from the latter by proteases of the furin family. Both TWEAK variants interact with high affinity with Fn14, an unusual small member of the TNF receptor family. TWEAK and Fn14 activate a variety of intracellular signaling pathways but regulation of TNF-induced cell death and stimulation of the classical and alternative NFκB pathway are certainly the best understood ones. Intriguingly, soluble and membrane TWEAK significantly differ in their ability to trigger these responses. While activation of the alternative NFκB pathway and enhancement of TNF-induced cell death are efficiently induced by both forms of TWEAK, membrane TWEAK has a much higher capacity than soluble TWEAK to stimulate the classical NFκB pathway. Importantly, soluble TWEAK gains a membrane TWEAK-like Fn14 stimulating activity upon oligomerization or artificial anchoring to the cell surface. On the example of NFκB signaling and enhancement of TNF-induced cell death, we summarize here protocols that allow the identification of signaling pathways/cellular responses that preferentially respond to membrane TWEAK. These protocols base either on the side-by-side analysis of soluble TWEAK and oligomerized or cell surface-anchorable TWEAK variants or on the use of transfectants expressing soluble and membrane TWEAK.