VanArsdale T L, Ware C F
Division of Biomedical Sciences, University of California, Riverside 92521.
J Immunol. 1994 Oct 1;153(7):3043-50.
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.
肿瘤坏死因子（TNF）是一种多能细胞因子，它通过两种不同的受体介导活性，这两种受体的分子量分别为55至60 kDa（CD120a，称为TNFR60）和75至80 kDa（CD120b，称为TNFR80）。这些受体在细胞外配体结合区域具有同源性；然而，细胞质结构域是不同的，并且缺乏任何固有的酶活性，这表明配体结合和随后的受体聚集导致活性信号分子与TNFRs结合。为了验证这一假设，我们通过免疫沉淀分离了TNFRs，并检查免疫复合物中是否存在相关的磷酸化蛋白和蛋白激酶活性。在预先用32PO4标记的U-937单核细胞系中，TNF诱导几种磷酸化蛋白与TNFR60结合，但不与TNFR80结合。TNFR60免疫复合物还含有一种TNF依赖性丝氨酸蛋白激酶活性，通过体外激酶测定检测到，该活性可磷酸化分子量为125、97、85和60 kDa的蛋白质，这些蛋白质的表观分子量与与TNFR60共沉淀的TNF诱导的磷酸化蛋白相似。丝氨酸蛋白激酶活性与TNFR60的结合迅速且依赖于TNF的浓度。分子量与125 kDa和97 kDa蛋白激酶底物相似的蛋白质似乎仅在U-937细胞暴露于TNF后才与TNFR60免疫复合物结合。与TNFR60相关的蛋白激酶活性受到星形孢菌素的抑制，但不受蛋白激酶A和C抑制剂HA-1004和H7的抑制。星形孢菌素大大增强了U-937细胞对TNF细胞毒性作用的敏感性。这些结果表明，一种丝氨酸蛋白激酶，以及可能的其他TNF依赖性TNFR60相关蛋白，可能参与介导通过TNFR60对配体结合的信号反应。
