The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protein kinases, was examined in the nervous system. DLK transcript expression in the nervous system was predominantly neuronal. DLK protein was present in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had differing characteristics. Cytosolic DLK existed in both a phosphorylated and dephosphorylated state; DLK associated with plasma membrane existed in the dephosphorylated state only. On nonreducing SDS-polyacrylamide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while membrane associated DLK migrated with an apparent M r 260,000. Similarly, DLK transiently expressed in COS 7 cells autophosphorylated in vivo and migrated at approximately 260 kDa when separated by nonreducing SDS-polyacrylamide gel electrophoresis. In cotransfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-⌬520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conclusion that DLK formed covalently associated homodimers in overexpressing COS 7 cells. In aggregating neuronal-glial cultures, depolarization of plasma membrane lead to dephosphorylation of DLK. Treatment of aggregates with 5 nM or 200 nM okadaic acid lead to a shift in electrophoretic mobility consistent with phosphorylation of DLK. Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulindependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.

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.

Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH 2 -terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.A large body of work has focused on signal transduction via protein kinases generically termed mitogen-activated protein kinases (MAPK) 1 that link a variety of extracellular signals to cellular responses as diverse as proliferation, differentiation, and apoptosis (reviewed in Refs. 1-3). Biochemical and genetic evidence has demonstrated that activation of a prototypical MAPK occurs through sequential activation of a series of upstream kinases: a serine/threonine MAPK kinase kinase (MAP-KKK) phosphorylates a dual specificity protein kinase (MAPKK or MKK or MEK) that in turn phosphorylates and activates a MAPK. Three groups of mammalian MAPKs and the upstream kinases and stimuli that activate them have been studied most extensively. These include the p42/p44 MAPK s (extracellular signal-regulated kinases, ERK1 and ERK2), that are generally activated by mitogens and differentiation inducing stimuli, the p46/p54 SAPK s (stress-activated protein kinases, SAPKs), and the p38 MAPK s. Stress-activated protein kinases were discovered as the principal c-Jun NH 2 -terminal kinases and therefore have also been termed JNKs. Distinct from ERK1 and ERK2, the SAPKs are predominantly activated by cell stress-inducing signals such as heat shock, ultraviolet irradiation, proinflammatory cytokines, hyperosmolarity, ischemia/reperfusion, and axonal injury.Like previously identified MAPK pathways in mammalian cells and yeast, the SAPK pathways were initially thought to lead in a lin...

Because the catalytic domain of dual leucine zipperbearing 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. While work in mammalian systems established the importance of the ERK pathway in signal transduction from RTKs, it has become clear from studies in yeast that multiple mammalian MAPK pathways exist in parallel (50). Using both genetic and biochemical approaches in mammalian cells, the components of several additional MAPK pathways have now been identified. Best characterized is the stress-activated protein kinase (SAPK) pathway. This pathway is thought to lead from the activated Rho subfamily small GTPases Rac1 and Cdc42Hs, to activation of the MAP kinase kinase kinase kinase, p65 PAK , to the MAP kinase kinase kinase, MEKK1, to the dual specificity MAP kinase kinase, MKK4/SEK1, and, finally, to activation of the MAP kinases p46/p54 SAPK . SAPKs were discovered as the principal c-Jun NH 2 -terminal phosphorylating kinases and therefore have also been termed JNKs (6). Distinct from the ERK cascade, the SAPK pathway is predominantly activated by stress-inducing signals such as heat shock, ultraviolet irradiation, anisomycin, proinflammatory cytokines (tumor necrosis factor ␣ and interleukin 1␤), and hyperosmolarity (7). Although G-protein-coupled receptors can signal through pathways leading to the activation of SAPK (8), the upstream signaling events by which the SAPK pathway becomes activated are largely unmapped.The mixed lineage kinase or MLK subfamily of protein kinases is a recently described subfamily of protein kinases that share two common structural features (9, 10). First, each has a distinctive kinase catalytic domain whose primary structure is hybrid between those found in serine/threonine and tyrosine protein kinases. Second, closely juxtaposed COOH-terminal to the catalytic domain, each MLK protein has a domain that is predicted to form two leucine/isoleucine zippers separated by a short spacer region. Additionally, each has both NH 2 -and COOH-terminal motifs suggestive of protein-protein interaction domains. Despite the hybrid structure of the catalytic domains, two members of the family (DLK and MLK3/SPRK) have been shown to exhibit serine/threonine-specific kinase autocatalytic activity in vitro (10 -12).

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.