DIRECT BINDING TO AND TYROSINE PHOSPHORYLATION OF THE ALPHA-SUBUNIT OF THE TYPE-I INTERFERON RECEPTOR BY P135(TYK2) TYROSINE KINASE
DIRECT BINDING TO AND TYROSINE PHOSPHORYLATION OF THE ALPHA-SUBUNIT OF THE TYPE-I INTERFERON RECEPTOR BY P135(TYK2) TYROSINE KINASE
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DOI：
10.1128/mcb.14.12.8133
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发表时间：
1994-12-01
影响因子：
5.3
通讯作者：
KROLEWSKI, J
中科院分区：
文献类型：
--
作者：
COLAMONICI, O;YAN, H;KROLEWSKI, J
I 型干扰素 (IFN) 与其受体结合可诱导多种蛋白质快速酪氨酸磷酸化，包括受体的 α 和 β 亚基、形成转录激活剂 ISGF3 α（Stat113、Stat84 和 Stat91）的多肽以及 p135(tyk2) 和 Jak-1 酪氨酸激酶。在本报告中，我们证明 I 型干扰素受体 (IFN-R) 的 α 亚基对应于先前克隆的受体亚基 cDNA 的产物，此外，p135(tyk2) 酪氨酸激酶直接结合该受体亚基并使酪氨酸磷酸化。编码 α 亚基胞质结构域不同区域的谷胱甘肽 S-转移酶 (GST) 融合蛋白可以结合人细胞裂解物中包含的 p135(tyk2)。 α亚基和 Tyk2 之间的关联通过抗 Tyk2 和抗磷酸酪氨酸抗体的免疫印迹以及使用体外激酶测定来证明。然后用编码组成型活性 Jak 家族酪氨酸激酶的重组杆状病毒进行类似的实验。在这种情况下，p135(tyk2)（而不是 Jak-1 或 Jak-2 蛋白）与 GST-IFN-R 蛋白结合，表明这两种蛋白之间的相互作用是直接且特异性的。我们还证明，来自 IFN α 处理的人类细胞或感染重组杆状病毒的昆虫细胞的提取物中的 Tyk2 可以以特定方式催化 GST-IFN-R 蛋白的体外磷酸化。 GST-IFN-R 蛋白的缺失突变体用于将结合位点和酪氨酸磷酸化位点定位于 α 亚基的 46 个氨基酸的近膜区域，该区域与其他细胞因子受体蛋白的功能相似区域显示出序列同源性。这些数据支持这样的假设：Tyk2 蛋白作为受体复合物的一部分发挥作用，启动响应 I 型干扰素的细胞内信号传导。
Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the alpha and beta subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3 alpha (Stat113, Stat84, and Stat91), and the p135(tyk2) and Jak-1 tyrosine kinases. In this report, we demonstrate that the alpha subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the p135(tyk2) tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the alpha subunit can bind the p135(tyk2) contained in human cell lysates. The association between the alpha subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, p135(tyk2), but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFN alpha-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the alpha subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.