TNF receptor signal transduction. Ligand-dependent stimulation of a serine protein kinase activity associated with (CD120a) TNFR60.
TNF is a pluripotent cytokine that mediates activities through two distinct receptors of 55 to 60 kDa (CD120a, known as TNFR60) and 75 to 80 kDa (CD120b, known as TNFR80). These receptors share homology in the extracellular ligand binding region; however, the cytoplasmic domains are distinct and lack any inherent enzymatic activity, which suggests that ligand binding and subsequent receptor clustering leads to the association of active signaling molecules with TNFRs. To test this hypothesis, we isolated TNFRs by immunoprecipitation and examined the immune complexes for the presence of associated phosphoproteins and protein kinase activity. In the U-937 monocytic cell line, prelabeled with 32PO4, TNF induces the association of several phosphoproteins with TNFR60, but not TNFR80. The TNFR60 immune complexes also contain a TNF-dependent serine protein kinase activity, which was detected by an in vitro kinase assay, that phosphorylates proteins of 125, 97, 85, and 60 kDa, which are of apparent molecular masses that are similar to those of TNF-induced phosphoproteins that coprecipitate with TNFR60. Association of serine protein kinase activity with TNFR60 is rapid and dependent on the concentration of TNF. Proteins of molecular mass similar to the 125- and 97-kDa protein kinase substrates seem to be associated with TNFR60 immune complexes only after exposure of U-937 cells to TNF. The TNFR60-associated protein kinase activity is inhibited by staurosporine, but not by the protein kinase A and C inhibitors, HA-1004 and H7. Staurosporine greatly enhanced the sensitivity of U-937 cells to the cytotoxic effect of TNF. These results suggest a serine protein kinase(s), and, possibly, other TNF-dependent TNFR60-associated proteins may be involved in mediating signals through TNFR60 in response to ligand binding.
VanArsdale
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Division of Biomedical Sciences, University of California, Riverside 92521.
Ware
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CF
D000470:Alkaloids / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D015398:Signal Transduction; D019311:Staurosporine
D000470:Alkaloids; D010750:Phosphoproteins; D018124:Receptors, Tumor Necrosis Factor; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine