title: Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Tomsig
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sohma
authors.forename: Carl E
authors.initials: CE
authors.lastname: Creutz
mesh_terms: 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
chemical_list: 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