Wada T, Takagi T, Yamaguchi Y, Kawase H, Hiramoto M, Ferdous A, Takayama M, Lee K A, Hurst H C, Handa H
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan.
Nucleic Acids Res. 1996 Mar 1;24(5):876-84. doi: 10.1093/nar/24.5.876.
We have developed a simple method to purify sequence-specific DNA-binding proteins directly from crude cell extracts by using DNA affinity latex beads. The method enabled us to purify not only DNA-binding proteins, but also their associated proteins. Using beads bearing the ATF/E4TF3 site from the adenovirus E4 gene promoter, a protein kinase activity was copurified with the ATF/E4TF3 family. We found that the kinase interacted with ATF1 in vitro efficiently. The kinase did not bind directly to DNA. The kinase mainly phosphorylated ATF1 on serine 36, which was one of target amino acids for casein kinase (CK) II. Biological features of the kinase were the same as those of CKII and an anti-CKII serum reacted with the kinase, indicating that the kinase was CKII. Moreover, it was clearly shown that one of CKII subunits, the CKII alpha protein bound to glutathione-S-transferase (GST) fusion ATF1 but not GST in vitro. It has been reported that a specific CKII inhibitor, 5,6-dichloro-1-beta-D-ribo-furanosylbenzimidazole (DRB) inhibits transcription by RNA polymerase II [Zandomeni et al., (1986) J. Biol. Chem. 261, 3414-3419]. Taken together, these results suggest that ATF/E4TF3 may recruit the CKII activity to a transcription initiation machinery and stimulate transcription.
我们开发了一种简单的方法，通过使用DNA亲和乳胶珠直接从粗细胞提取物中纯化序列特异性DNA结合蛋白。该方法不仅使我们能够纯化DNA结合蛋白，还能纯化其相关蛋白。使用带有腺病毒E4基因启动子的ATF/E4TF3位点的珠子，一种蛋白激酶活性与ATF/E4TF3家族一起被共纯化。我们发现该激酶在体外能有效地与ATF1相互作用。该激酶不直接与DNA结合。该激酶主要在丝氨酸36处磷酸化ATF1，丝氨酸36是酪蛋白激酶（CK）II的靶氨基酸之一。该激酶的生物学特性与CKII相同，并且一种抗CKII血清能与该激酶发生反应，表明该激酶就是CKII。此外，清楚地表明，CKII的一个亚基，即CKIIα蛋白在体外能与谷胱甘肽-S-转移酶（GST）融合的ATF1结合，但不与GST结合。据报道，一种特异性的CKII抑制剂，5,6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB）可抑制RNA聚合酶II的转录[赞多梅尼等人，（1986年）《生物化学杂志》261，3414 - 3419]。综上所述，这些结果表明ATF/E4TF3可能将CKII活性募集到转录起始机制中并刺激转录。
