Wang Y, Segall J E
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochem Biophys Res Commun. 1998 Mar 6;244(1):149-55. doi: 10.1006/bbrc.1997.8118.
A polyclonal antibody against a MAP kinase (DdERK2) in Dictyostelium has been made and used to study DdERK2 activation and localization. The activation of DdERK2 by the chemoattractants cAMP and folate is rapid and transient. Its activity peaks between 15 and 60 seconds after cAMP stimulation and declines to basal levels after 5 minutes. In parallel with the DdERK2 activation is the appearance of a higher mobility band on Western blots. An antibody specific for activated MAP kinase shows that only the shifted band is tyrosine phosphorylated, suggesting that it is the active form. Both unstimulated and stimulated DdERK2 are soluble. In vitro phosphorylation with cell lysate supernatants or immunoprecipitates demonstrates the presence of several potential substrates, as identified by SDS-PAGE with mobility corresponding to molecular weights of 150, 25, and 19 kDa. Furthermore, immunoprecipitation studies suggest that these substrates are in a complex with DdERK2. These data suggest that DdERK2 works via cytoplasmic proteins to mediate signaling responses in Dictyostelium.
已制备出一种针对盘基网柄菌中一种丝裂原活化蛋白激酶（DdERK2）的多克隆抗体，并用于研究DdERK2的激活和定位。趋化因子cAMP和叶酸对DdERK2的激活迅速且短暂。其活性在cAMP刺激后15至60秒达到峰值，并在5分钟后降至基础水平。与DdERK2激活同时出现的是，蛋白质免疫印迹上出现了一条迁移率更高的条带。一种针对活化丝裂原活化蛋白激酶的特异性抗体表明，只有迁移的条带发生了酪氨酸磷酸化，这表明它是活性形式。未刺激和刺激后的DdERK2均为可溶性。用细胞裂解物上清液或免疫沉淀物进行的体外磷酸化实验表明，存在几种潜在底物，通过SDS-PAGE鉴定，其迁移率对应于分子量为150、25和19 kDa的蛋白质。此外，免疫沉淀研究表明，这些底物与DdERK2形成复合物。这些数据表明，DdERK2通过细胞质蛋白发挥作用，介导盘基网柄菌中的信号转导反应。
