Fan G, Merritt S E, Kortenjann M, Shaw P E, Holzman L B
Division of Nephrology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676, USA.
J Biol Chem. 1996 Oct 4;271(40):24788-93. doi: 10.1074/jbc.271.40.24788.
Because the catalytic domain of dual leucine zipper-bearing kinase (DLK) bears sequence similarity to members of the mitogen-activated protein (MAP) kinase kinase kinase subfamily, this protein kinase was investigated for its ability to activate MAP kinase pathways. When transiently transfected and overexpressed in either COS 7 cells or NIH3T3 cells, wild type DLK potently activated p46(SAPK) (SAPK/JNK) but had no detectable effect in activating p42/44(MAPK). DLK also activated p38(mapk) when overexpressed in NIH3T3 cells. A catalytically inactive point mutant of DLK had no effect in these experiments. Consistent with its specificity in activating SAPK, DLK activated Elk-1 but not Sap1a-mediated transcription. In NIH3T3 cells, activation of SAPK by v-Src was markedly attenuated by coexpression of K185A, a catalytically inactive mutant of DLK, suggesting that this mutant could function in a dominant negative fashion in a pathway that leads from v-Src to SAPKs. In a series of co-transfection experiments, activation of p46(SAPK) by DLK was not inhibited by dominant negative mutants of Rac1 and Cdc42Hs, PAK65-R, or PAK65-A, but was attenuated by MEKK1(K432M). DLK(K185A) did not inhibit the ability of constitutively active MEKK1 to activate SAPK. Moreover, K185A significantly inhibited the activation of SAPK by constitutively active V-12 Rac1 and V-12 Cdc42Hs. These results suggest that DLK lies distal to Rac1 and/or Cdc42Hs but proximal to MEKK1 in a pathway leading from v-Src to SAPKs activation.
由于双亮氨酸拉链激酶（DLK）的催化结构域与丝裂原活化蛋白（MAP）激酶激酶激酶亚家族成员的序列相似，因此对该蛋白激酶激活MAP激酶途径的能力进行了研究。当在COS 7细胞或NIH3T3细胞中瞬时转染并过表达时，野生型DLK能有效激活p46（SAPK）（SAPK/JNK），但对激活p42/44（MAPK）没有可检测到的作用。当在NIH3T3细胞中过表达时，DLK也能激活p38（mapk）。DLK的催化失活点突变体在这些实验中没有作用。与其激活SAPK的特异性一致，DLK激活了Elk-1，但没有激活Sap1a介导的转录。在NIH3T3细胞中，v-Src对SAPK的激活被DLK的催化失活突变体K185A的共表达显著减弱，这表明该突变体可以在从v-Src到SAPKs的途径中以显性负性方式发挥作用。在一系列共转染实验中，Rac1和Cdc42Hs、PAK65-R或PAK65-A的显性负性突变体没有抑制DLK对p46（SAPK）的激活，但MEKK1（K432M）使其减弱。DLK（K185A）没有抑制组成型活性MEKK1激活SAPK的能力。此外，K185A显著抑制了组成型活性V-12 Rac1和V-12 Cdc42Hs对SAPK的激活。这些结果表明，在从v-Src到SAPKs激活的途径中，DLK位于Rac1和/或Cdc42Hs的下游，但在MEKK1的上游。
