Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine

Abstract

The role of copines in regulating signalling from the TNF-α (tumour necrosis factor-α) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-κB (nuclear factor-κB) by TNF-α. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-α-dependent activation of NF-κB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-α was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-α signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-α-dependent degradation of IκB, a regulator of NF-κB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IκB through the ubiquitin ligase enzyme complex SCFβTrCP. Therefore the copine dominant-negative construct might inhibit TNF-α signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa.