Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.
The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) 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-kappaB (nuclear factor-kappaB) by TNF-alpha. 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-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. 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 IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha 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.
Tomsig
Jose Luis
JL
Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
Sohma
Hitoshi
H
Creutz
Carl E
CE
D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002352:Carrier Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; C110203:copine; D002118:Calcium