A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in 'reverse signalling'

We have identified a putative signalling feature of the cytoplasmic domains of the tumour necrosis factor (TNF) family members based on available amin o acid sequence data. A casein kinase I (CKI) consensus sequence is conserv ed in the cytoplasmic domain of six of 15 members of the type II integral m embrane TNF ligand family. We examined the phosphorylation state of transme mbrane tumour necrosis factor-alpha (mTNF) with [P-32]orthophosphate labell ing and irt vitro kinase assays,in lipopolysaccharide-stimulated RAW264.7 c ells. A dimeric form of the type I soluble TNF receptor (sTNFR) was found t o dephosphorylate mTNF. This effect could be prevented by treatment with ph osphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that h ad been dephosphorylated by sTNFR ligation lit vivo, and this was less effe ctive if phosphatase inhibitors had been used to prevent mTNF dephosphoryla tion. A mutated form of mTNF, lacking the CKI recognition site, cannot be p hosphorylated by the enzyme. Binding of sTNFR to mTNF induced an increase i n intracellular calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phospho rylated in other TNF ligand members, and that it represents a new insight i nto the mechanism of 'reverse signalling' in this cytokine family.