Northwood I C, Gonzalez F A, Wartmann M, Raden D L, Davis R J
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605.
J Biol Chem. 1991 Aug 15;266(23):15266-76.
A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.
已报道一种生长因子刺激的蛋白激酶活性，其可使表皮生长因子（EGF）受体的苏氨酸669位点发生磷酸化（Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828 - 10835）。阴离子交换色谱显示这种蛋白激酶活性由两种酶构成。从柱上洗脱下来的第一个活性峰对应微管相关蛋白2（MAP2）激酶。然而，第二个活性峰是一种不同的酶。我们在此展示了通过连续离子交换色谱从人肿瘤KB细胞中纯化该酶的过程。通过聚丙烯酰胺凝胶电泳和银染法，将分离得到的蛋白激酶鉴定为一种46 kDa的蛋白。凝胶过滤色谱表明该酶以单体状态发挥功能。在22℃下，使用基于EGF受体一级序列（KREL VEPLT669PSGEAPNQALLR）的合成肽底物对纯化后的酶进行动力学分析。ATP的表观米氏常数（Km(app)）为40±5μM（平均值±标准差，n = 3）。未发现GTP是纯化后酶的底物。合成肽底物的表观米氏常数（Km(app)）为260±40μM（平均值±标准差，n = 3）。分离得到的蛋白激酶的最大反应速度（Vmax(app)）测定为400 - 900 nmol/mg/min。纯化后的酶被命名为EGF受体苏氨酸669（ERT）激酶。MAP2激酶和ERT激酶可能是导致在培养细胞中观察到的EGF受体苏氨酸669位点磷酸化的原因。生长因子对蛋白激酶活性的显著刺激表明这些酶在信号转导过程中可能具有重要功能。
