Dang A, Frost J A, Cobb M H
University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1998 Jul 31;273(31):19909-13. doi: 10.1074/jbc.273.31.19909.
MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.
MEK1和MEK2含有一段富含脯氨酸的插入序列，这在其他任何已知的MEK（丝裂原活化蛋白/细胞外信号调节激酶激酶）家族成员中都不存在。我们研究了去除MEK1多聚脯氨酸插入序列对MEK活性、其与Raf的结合以及其在细胞中激活ERK的能力的影响。去除该插入序列对MEK1的活性或其与Raf-1的结合能力均无影响。无论插入序列是否存在，野生型和组成型活性MEK1均能与Raf-1进行共免疫沉淀。去除插入序列并未降低细胞中表皮生长因子（EGF）或活化的Raf对MEK1的激活作用。富含脯氨酸的插入序列增强了原本活性相当的MEK1蛋白在哺乳动物细胞中调节内源性ERK的能力。缺乏插入序列的组成型活性MEK1或ERK2的过表达可弥补MEK1缺失突变体在体内较弱的活性。在细胞中表达该插入序列可降低EGF对ERK的激活作用。我们得出结论，富含脯氨酸的插入序列不是MEK与Raf相互作用的位点，并且多聚脯氨酸插入序列是其在细胞中有效激活下游ERK所必需的。
