Müller E, Scheidtmann K H
Department of Molecular Genetics, University of Bonn, Germany.
Oncogene. 1995 Mar 16;10(6):1175-85.
In SV40-transformed or infected rat cells phosphorylation of the tumor suppressor protein p53 is enhanced due to activation of kinases. At least three different kinases can be co-precipitated with p53-large T (LT) immune complexes, casein kinase II representing the major activity, a cyclin dependent kinase (Cdk), and a kinase which appears to be specifically activated by LT (E Müller, B Boldyreff and KH Scheidtmann, Oncogene 8: 2193-2205, 1993). In this paper we describe the purification and identification of the LT-activated kinase that phosphorylates a site adjacent to the Cdk site in rat p53. To monitor the activity a synthetic peptide was used containing glutamic acid at the position of Ser-313, thus mimicking a phosphorylated Cdk site. With a combination of Mono Q chromatography and subsequent affinity chromatographies with p13suc1 and a p53-fragment as ligands a 42 kDa protein kinase was purified to near homogeneity from SV40-transformed rat cells. This kinase phosphorylated both the peptide substrate and the native rat p53. Interestingly, phosphorylation of the specific site seemed to depend on prior phosphorylation of the Cdk site. On the other hand, the kinase seemed to be activated by LT, as the activity towards the peptide substrate was significantly higher in extracts from wild-type LT-transformed cells than from normal or mutant LT-transformed cells. This activation was not restricted to rat cells but occurred in SV40-transformed mouse and infected monkey cells as well. Phosphorylation of the specific site by LT-activated kinase was not dependent on the presence of LT in vitro suggesting that activation of the LT-activated kinase is probably indirect rather than through direct interaction with LT. Cell cycle studies revealed that the LT-activated kinase is cell cycle regulated, since its activity was not detectable in M phase but increased during G1 phase after which it remained relatively constant.
在SV40转化或感染的大鼠细胞中，由于激酶的激活，肿瘤抑制蛋白p53的磷酸化作用增强。至少有三种不同的激酶可与p53大T（LT）免疫复合物共沉淀，其中酪蛋白激酶II代表主要活性，一种细胞周期蛋白依赖性激酶（Cdk），以及一种似乎被LT特异性激活的激酶（E·米勒、B·博尔迪列夫和KH·谢特曼，《癌基因》8：2193 - 2205，1993）。在本文中，我们描述了对LT激活的激酶的纯化和鉴定，该激酶使大鼠p53中与Cdk位点相邻的一个位点发生磷酸化。为监测该活性，使用了一种在Ser - 313位置含有谷氨酸的合成肽，从而模拟磷酸化的Cdk位点。通过Mono Q色谱法以及随后以p13suc1和一个p53片段作为配体的亲和色谱法相结合，从SV40转化的大鼠细胞中纯化出一种42 kDa的蛋白激酶，纯度接近均一。该激酶使肽底物和天然大鼠p53都发生了磷酸化。有趣的是，特定位点的磷酸化似乎依赖于Cdk位点的预先磷酸化。另一方面，该激酶似乎被LT激活，因为野生型LT转化细胞提取物中对肽底物的活性明显高于正常或突变LT转化细胞。这种激活并不局限于大鼠细胞，在SV40转化的小鼠细胞和感染的猴细胞中也会发生。LT激活的激酶对特定位点的磷酸化在体外不依赖于LT的存在，这表明LT激活的激酶的激活可能是间接的，而非通过与LT的直接相互作用。细胞周期研究表明，LT激活的激酶受细胞周期调控，因为在M期检测不到其活性，但在G1期增加，此后保持相对恒定。
