Identification of Structural and Functional Domains in Mixed Lineage Kinase Dual Leucine Zipper-bearing Kinase Required for Complex Formation and Stress-activated Protein Kinase Activation

Abstract

Accumulating evidence suggests that mitogen-activated protein kinase signaling pathways form modular signaling complexes. Because the mixed lineage kinase dual leucine zipper-bearing kinase (DLK) is a large modular protein, structure-function analysis was undertaken to examine the role of DLK domains in macromolecular complex formation. DLK mutants were used to demonstrate that a DLK leucine zipper-leucine zipper interaction is necessary for DLK dimerization and to show that DLK dimerization mediated by the leucine zipper domain is prerequisite for DLK activity and subsequent activation of stress-activated protein kinase (SAPK). Heterologous mixed lineage kinase family members can be co-immunoprecipitated. However, the DLK leucine zipper domain interacted specifically only with the DLK leucine zipper domain; in contrast, DLK NH 2 -terminal region was sufficient to co-immunoprecipitate leucine zipper kinase and DLK. DLK has been shown to associate with the putative scaffold protein JIP1. This association occurred through the DLK NH 2 -terminal region and occurred independently of DLK catalytic activity. Although the DLK NH 2 -terminal region associated directly with JIP-1, this region did not interact directly with either DLK or leucine zipper kinase. Therefore, DLK may interact with heterologous mixed lineage kinase proteins via intermediary proteins. The NH 2 -terminal region of overexpressed DLK was required for activation of SAPK. These results provide evidence that protein complex formation is required for signal transduction from DLK to SAPK.

Inhibition of membrane depolarisation-induced transcriptional activity of cyclic AMP response element binding protein (CREB) by the dual-leucine-zipper-bearing kinase in a pancreatic islet beta cell line

“…In aggregating neuronal-glial cultures, depolarisation of plasma membrane led to dephosphorylation of DLK that was blocked by cyclosporin A, suggesting that the phosphorylation state of DLK is regulated by membrane depolarisation via calcineurin [19]. Consistent with the view that lack of autophosphorylation renders DLK enzymically inactive [34], DLK kinase activity was shown in the present study to be enhanced by cyclosporin A and tacrolimus, which are known to inhibit calcineurin phosphatase activity with high potency also in pancreatic islet beta cells [4,16,17,29]. On the other hand, overexpression of calcineurin decreased the inhibition by DLK of membrane depolarisation-induced CRE/CREB transcriptional activity.…”

“…It is assumed that signals promoting the dissociation of DLK from JIP lead to homodimerisation of DLK via its leucine zipper domains, followed by its autophosphorylation and activation [35][36][37]. Thus the overexpression of DLK also results in its activation [34]. The present study demonstrates that DLK is expressed in the insulin-producing pancreatic islet beta cell line HIT and in primary mouse islets including beta cells.…”

“…As such, DLK phosphorylates and activates the dualspecificity MAPKK MKK7 and MKK4, which in turn phosphorylate and activate the MAPK JNK [26,33,34]. Cterminal of the kinase domain DLK has two leucine zipper motifs that mediate protein dimerisation by forming coiledcoil structures [18].…”

“…Cterminal of the kinase domain DLK has two leucine zipper motifs that mediate protein dimerisation by forming coiledcoil structures [18]. However, within the cell and under basal conditions, DLK is associated with the scaffold proteins JNK interacting protein (JIP) [34][35][36] and Plenty of SH3 [36], which assemble and facilitate the activation of the DLK-dependent JNK module. JIP maintains DLK in a monomeric, unphosphorylated, inactive state [35,36].…”

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

“…[28][29][30][31] In developmental studies, it has been reported that DLK is expressed in E10 and involves in late neuronal development. [32][33][34][35][36] However, in mouse ES cells or in developmental stages before E10, the functional role(s) of DLK are unknown.…”