Watts A D, Hunt N H, Wanigasekara Y, Bloomfield G, Wallach D, Roufogalis B D, Chaudhri G
Departments of Pathology, University of Sydney, NSW 2006, Australia.
EMBO J. 1999 Apr 15;18(8):2119-26. doi: 10.1093/emboj/18.8.2119.
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.
基于现有的氨基酸序列数据，我们已经确定了肿瘤坏死因子（TNF）家族成员胞质结构域的一个假定信号特征。酪蛋白激酶I（CKI）共有序列在II型整合膜TNF配体家族15个成员中的6个成员的胞质结构域中保守。我们用[32P]正磷酸盐标记和体外激酶测定法，在脂多糖刺激的RAW264.7细胞中检测了跨膜肿瘤坏死因子-α（mTNF）的磷酸化状态。发现I型可溶性TNF受体（sTNFR）的二聚体形式可使mTNF去磷酸化。用磷酸酶抑制剂处理可阻止这种作用。重组CKI能够使体内因sTNFR连接而去磷酸化的mTNF磷酸化，如果使用磷酸酶抑制剂来阻止mTNF去磷酸化，这种效果则较差。缺乏CKI识别位点的mTNF突变形式不能被该酶磷酸化。sTNFR与mTNF的结合导致RAW264.7细胞内钙水平升高，这意味着存在相关的信号通路。我们预测，这个CKI基序在其他TNF配体成员中被磷酸化，并且它代表了对这个细胞因子家族中“反向信号传导”机制的新见解。
