JSAP1, a novel Jun N-terminal protein kinase (JNK)-binding protein that functions as a scaffold factor in the JNK signaling pathway

The major components of the mitogen-activated protein kinase (MAPK) cascade s are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid pr ogress in identifying members of MARK cascades suggests that a number of su ch signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. He re, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MARK as the bait. Of the mammalia n MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38 alpha), JSAP1 preferentiall y coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAP KKK, which are involved in the JNK cascades. The regions of JSAP1 that boun d JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also b ound JSAP1 in vitro, suggesting that these interactions are direct. In cont rast, only the activated form of SEK1 associated with JSAP1 in cotransfecte d COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indir ectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated wit h MEK1 MAPKK and Raf-l MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-l do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enha ncement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in t he enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also di scuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.