Identification of a protein kinase associated with the cytoplasmic domain of the p60 tumor necrosis factor receptor.
Tumor necrosis factor (TNF) has been shown to bind two distinct receptors, designated p60 and p80, with high affinity, resulting, within minutes, in phosphorylation of several proteins. The receptors themselves do not exhibit protein kinase activity nor have any associated proteins been identified. We employed the glutathione-S-transferase (GST) fusion protein system consisting of the cytoplasmic domain of p60 (GST-p60CD delta 1) as a probe to help us identify receptor-associated proteins from human histiocytic lymphoma U-937 cells. We found that a protein of approximately 52 kDa (pp52) bound to GST-p60CD delta 1 from [35S]methionine- and 32P-labeled cells. The associated protein was phosphorylated on serine and threonine residues. Furthermore, we identified serine/threonine kinase activity associated with p60CD delta 1 that required either Mn2+ or Mg2+ for optimal activity. The preferred substrates for this kinase, in addition to p60CD delta 1, included casein and histone H1, but not histone H2B, myelin basic protein, enolase, or the cytoplasmic domain of p80. As was the case in U-937 cells, p60CD delta 1-associated kinase activity was also identified in human breast adenocarcinoma MCF-7 cells and human foreskin fibroblasts. TNF stimulation of MCF-7 and foreskin fibroblasts for 5-15 min induced approximately 50 and 240% increases in phosphorylation of p60CD delta 1, respectively. Thus, our results provide the first evidence for protein kinase activity that is specifically associated with the cytoplasmic domain of the p60 form of the TNF receptor and causes its phosphorylation. This p60 TNF receptor-associated protein and the associated kinase described here are referred to as p60-TRAP and p60-TRAK, respectively.
Darnay
B G
BG
Department of Clinical Immunology and Biological Therapy, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Reddy
S A
SA
Aggarwal
B B
BB
D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017931:DNA Primers; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D011494:Protein Kinases