We have identified a putative signalling feature of the cytoplasmic domains of the tumour
necrosis factor (TNF) family members based on available amino acid sequence data. A
casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of six of 15 members of the type II integral membrane TNF
ligand family. We examined the phosphorylation state of transmembrane tumour
necrosis factor-alpha (mTNF) with [32P]
orthophosphate labelling and in vitro
kinase assays, in
lipopolysaccharide-stimulated RAW264.7 cells. A dimeric form of the type I soluble
TNF receptor (sTNFR) was found to dephosphorylate mTNF. This effect could be prevented by treatment with
phosphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that had been dephosphorylated by sTNFR
ligation in vivo, and this was less effective if
phosphatase inhibitors had been used to prevent mTNF dephosphorylation. A mutated form of mTNF, lacking the CKI recognition site, cannot be phosphorylated by the
enzyme. Binding of sTNFR to mTNF induced an increase in intracellular
calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phosphorylated in other TNF
ligand members, and that it represents a new insight into the mechanism of 'reverse signalling' in this
cytokine family.