Bird T A, Sleath P R, deRoos P C, Dower S K, Virca G D
Department of Biochemistry, Immunex Corporation, Seattle, Washington 98101.
J Biol Chem. 1991 Nov 25;266(33):22661-70.
In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.
在本研究中，我们描述了一种蛋白激酶的激活情况，该激酶可使一种肽（T669）磷酸化，T669由表皮生长因子受体的663 - 681位氨基酸组成，包含磷酸受体位点Pro - Leu - Thr669 - Pro。在人表皮样癌细胞系KB中，添加白细胞介素-1（IL-1）后15 - 30分钟，胞质提取物中的T669激酶活性达到峰值（与基础水平相比高达15倍），且与受体占有率密切相关，IL-1α的半数有效浓度约为100 pM。IL-1处理在选定的成纤维细胞系、肝癌细胞系HepG2和鼠胸腺瘤EL4 6.1中不同程度地提高了T669激酶活性。IL-1受体阴性的EL4变体以及B细胞系70Z/3、CB23和RPMI 1788未出现这种反应。除70Z/3外，所有细胞系在用蛋白激酶C激活剂佛波酯肉豆蔻酸酯乙酸盐和/或表皮生长因子处理后，T669激酶水平均升高。这一发现与之前的一项研究一致（Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828 - 10835）。蛋白激酶A的激活剂不能模拟IL-1刺激T669激酶活性的能力，蛋白激酶C抑制剂星形孢菌素也不能消除IL-1的作用。来自IL-1刺激的KB细胞的T669激酶活性通过离子交换、疏水相互作用和尺寸排阻色谱法进行了部分纯化。部分纯化的酶可使髓鞘碱性蛋白磷酸化，髓鞘碱性蛋白是微管相关蛋白-2激酶（MAP-2激酶）的特征性底物，还可使来自RNA聚合酶II的肽Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4磷酸化。对色谱级分进行蛋白质印迹分析表明，T669激酶活性与两种分别为43和45千道尔顿的蛋白质相对应，这两种蛋白质与针对大鼠细胞外信号调节激酶-1/微管相关蛋白-2激酶的肽序列产生的抗体发生交叉反应。T669激酶活性严重依赖于磷酸酶抑制剂的存在。由于从[32P]磷酸盐标记的细胞中免疫沉淀的43 kDa和45 kDa蛋白质在用IL-1短暂处理后，其丝氨酸、苏氨酸和酪氨酸磷酸化水平均显著增加，我们得出结论，IL-1通过改变其磷酸化水平来调节这些细胞外信号调节激酶/微管相关蛋白-2激酶的活性。
