Murata T, Noguchi P D, Puri R K
Laboratory of Molecular Tumor Biology, Food and Drug Administration, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Dec 22;270(51):30829-36. doi: 10.1074/jbc.270.51.30829.
We have previously reported on the expression of interleukin-4 receptors (IL-4R) on many human epithelial cancer cells; however, the binding characteristics, structure, function, and signal transduction through the IL-4R in cancer cells is not known. IL-4 binding characteristics were determined in human colon carcinoma cell lines by a 125I-IL-4 binding assay, which demonstrated that the HT-29 and WiDr colon cancer cell lines expressed high affinity IL-4R (Kd = 200 pM). Cross-linking experiments revealed a major band of 140 kDa and a broad band at 70 kDa. While the common gamma chain of IL-2R is associated with IL-4R in immune cells and is similar in size to the 70-kDa protein, this chain was not expressed in these colon cancer cells. Interestingly, IL-13, which has many functions similar to IL-4, inhibited 125I-IL-4 binding to both the 140- and 70-kDa molecules. Next, we investigated the mechanism of IL-4-induced signal transduction in colon cancer cells. After stimulation with IL-4, a 170-kDa band was primarily phosphorylated within 1 min of exposure and was identified as insulin receptor substrate-1. In addition, by immunoprecipitation assay, three other phosphorylated bands were identified as JAK1, JAK2, and Tyk2 tyrosine kinases. The phosphorylation of JAK1 and JAK2 was induced by IL-4 stimulation; however, Tyk2 was constitutively phosphorylated, and IL-4 treatment further augmented this phosphorylation. The kinetics and in vitro kinase assays demonstrated that JAK1, JAK2, and Tyk2 were phosphorylated within minutes and that JAK1 and JAK2 were activated after IL-4 exposure. Contrary to observations in immune cells. JAK3 mRNA was neither detected in colon cancer cells nor did IL-4 treatment cause phosphorylation of JAK3. These data indicate that in colon carcinoma cells JAK1, JAK2, Tyk2, and insulin receptor substrate-1 are phosphorylated after IL-4 stimulation. In addition, as is the case in lymphoid cells, IL-4 activated and phosphorylated signal transducers and activators of transcription (IL-4-STAT or STAT-6) protein in both colon cancer cell lines. These results indicate that the IL-4R complex is composed of different subunits in different tissues and shares a component with the IL-13R complex. In addition, we demonstrate for the first time that like its family members (e.g. IL-3 and GM-CSF), IL-4 can phosphorylate and activate JAK-2 kinase.
我们之前曾报道过许多人类上皮癌细胞上白细胞介素-4受体（IL-4R）的表达情况；然而，癌细胞中IL-4R的结合特性、结构、功能及信号转导尚不清楚。通过125I-IL-4结合试验测定了人结肠癌细胞系中的IL-4结合特性，结果表明HT-29和WiDr结肠癌细胞系表达高亲和力IL-4R（解离常数Kd = 200 pM）。交联实验显示一条140 kDa的主要条带和一条70 kDa的宽带。虽然IL-2R的共同γ链在免疫细胞中与IL-4R相关联且大小与70 kDa蛋白相似，但该链在这些结肠癌细胞中未表达。有趣的是，与IL-4具有许多相似功能的IL-13抑制125I-IL-4与140 kDa和70 kDa分子的结合。接下来，我们研究了IL-4诱导的结肠癌细胞信号转导机制。用IL-4刺激后，一条170 kDa的条带在暴露1分钟内主要发生磷酸化，被鉴定为胰岛素受体底物-1。此外，通过免疫沉淀试验，另外三条磷酸化条带被鉴定为JAK1、JAK2和Tyk2酪氨酸激酶。JAK1和JAK2的磷酸化由IL-4刺激诱导；然而，Tyk2是组成型磷酸化的，IL-4处理进一步增强了这种磷酸化。动力学和体外激酶试验表明JAK1、JAK2和Tyk2在数分钟内发生磷酸化，且IL-4暴露后JAK1和JAK2被激活。与免疫细胞中的观察结果相反，在结肠癌细胞中未检测到JAK3 mRNA，IL-4处理也未导致JAK3磷酸化。这些数据表明在结肠癌细胞中，IL-4刺激后JAK1、JAK2、Tyk2及胰岛素受体底物-1发生磷酸化。此外，与淋巴细胞情况相同，IL-4在两种结肠癌细胞系中激活并磷酸化信号转导子和转录激活子（IL-4-STAT或STAT-6）蛋白。这些结果表明IL-4R复合物在不同组织中由不同亚基组成，并与IL-13R复合物有一个共同成分。此外，我们首次证明，与它的家族成员（如IL-3和GM-CSF）一样，IL-4能磷酸化并激活JAK-2激酶。
