Baier L J, Shors T, Shors S T, Jacobs B L
Department of Microbiology, Arizona State University, Tempe 85287-2701.
Nucleic Acids Res. 1993 Oct 11;21(20):4830-5. doi: 10.1093/nar/21.20.4830.
The mouse TIK protein, a serine/threonine kinase, was originally isolated from a murine pre-B cell expression library by its ability to bind anti-phosphotyrosine antibodies (Icely et al., J. Biol. Chem. 266, 16073-16077, 1991). The 67 kDa protein was found to have an associated autophosphorylation activity when incubated with ATP. Our results show that TIK is actually the mouse interferon-induced, dsRNA-dependent protein kinase, PKR. We demonstrate that the TIK message is interferon-inducible in mouse L-cells and in vitro transcription and translation of the TIK cDNA produces a protein that is capable of binding double-stranded RNA. The in vitro synthesized TIK protein migrated as a 65 kDa protein on SDS-PAGE when incubated with ATP, but migrated as a 60 kDa protein when incubated with an inhibitor of PKR, 2-aminopurine. We further show that proteolytic digestion of TIK with Staphylococcus aureus V8 protease results in a cleavage pattern identical to that obtained by V8 digestion of authentic PKR. Antiserum to TIK specifically recognized PKR. Cloned TIK had inhibitory activity for replication of EMCV but not VSV. From these observations we conclude that TIK kinase is the mouse interferon-induced, double-stranded RNA-dependent kinase, PKR.
小鼠TIK蛋白是一种丝氨酸/苏氨酸激酶，最初是从鼠前B细胞表达文库中通过其结合抗磷酸酪氨酸抗体的能力分离得到的（Icely等人，《生物化学杂志》266，16073 - 16077，1991）。发现该67 kDa蛋白与ATP孵育时具有相关的自磷酸化活性。我们的结果表明，TIK实际上就是小鼠干扰素诱导的、双链RNA依赖性蛋白激酶PKR。我们证明，TIK信使RNA在小鼠L细胞中可被干扰素诱导，并且TIK cDNA的体外转录和翻译产生一种能够结合双链RNA的蛋白。体外合成的TIK蛋白与ATP孵育时在SDS - PAGE上迁移为65 kDa的蛋白，但与PKR抑制剂2 - 氨基嘌呤孵育时迁移为60 kDa的蛋白。我们进一步表明，用金黄色葡萄球菌V8蛋白酶对TIK进行蛋白水解消化产生的裂解模式与对天然PKR进行V8消化所得到的模式相同。TIK抗血清能特异性识别PKR。克隆的TIK对脑心肌炎病毒（EMCV）的复制具有抑制活性，但对水泡性口炎病毒（VSV）没有抑制活性。基于这些观察结果，我们得出结论，TIK激酶就是小鼠干扰素诱导的、双链RNA依赖性激酶PKR。
