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.
Highlights
Among the MAPKs, the stress-activated protein kinases (SAPKs or JNKs) are predominantly activated by cell stressinducing signals, such as heat shock, ultraviolet irradiation, proinflammatory cytokines, hyperosmolarity, ischemia/reperfusion, and axonal injury
Activation of a prototypical MAPK occurs through sequential activation of a series of upstream kinases: a serine/threonine MAPK kinase kinase (MAPKKK) phosphorylates a dual specificity protein kinase (MAPK kinase) that in turn phosphorylates and activates a MAPK
With the identification of two MAPK kinases (MKK4/ SEK1 and MKK7/JNKK2) many MAPKKKs (four mitogenactivated protein kinase/extracellular signal regulated kinase kinase kinases 1, five mixed lineage kinases (MLKs), ASK1, Tpl-2, and TAK1) and multiple additional MAPKKK kinases that appear to lie in pathways proximal to the SAPKs, it is clear that the organization, regulation, and function of these protein kinase pathways remain incompletely understood
Summary
Among the MAPKs, the stress-activated protein kinases (SAPKs or JNKs) are predominantly activated by cell stressinducing signals, such as heat shock, ultraviolet irradiation, proinflammatory cytokines, hyperosmolarity, ischemia/reperfusion, and axonal injury. Distinct MAPK pathways that respond to specific stimuli and effect unique cellular responses may employ subsets of identical protein kinases within the same cell. Studies in yeast have provided evidence that MAP kinase pathways are assembled from a unique combination of protein kinases into distinct protein complexes or modules (6, 7). Identification of JIP-1 as a scaffold protein that interacts in a specific fashion with members of the MLK family of MAPKKKs, with MKK7 but not MKK4, and with JNKs first established that mammalian cells organize SAPK pathways into modules in a fashion similar to yeast (13). Closely juxtaposed COOH-terminal to the catalytic domain, each MLK protein has two leucine/isoleucine zippers separated by a short spacer region Despite these common features, MLK family members are likely to have diverse biological behavior. This is predicted by comparison of structure that shows that MLKs 1, 2, and 3 form
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