The Mixed Lineage Kinase DLK Utilizes MKK7 and Not MKK4 as Substrate

Abstract

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...

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

“…DLK had no effect on basal activity (results not shown). To test whether the kinase activity of DLK is required for the effect of DLK, a DLK kinase-dead mutant was employed in which the exchange of Lys185 to Ala within the ATP-binding domain abolishes DLK kinase activity [19,26]. As shown in Fig.…”

“…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].…”

Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper

“…4) (Dorow et al 1993(Dorow et al , 1995Gallo et al 1994;Ing et al 1994;Katoh et al 1995). The structural features of MLK proteins have provided clues to their function and support their role as important signal transducers for the JNK pathway, conveying signals from upstream GTPases to downstream JNKKs, (Rana et al 1996;Teramoto et al 1996;Tibbles et al 1996;Hirai et al 1997;Merritt et al 1999).…”

Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells

“…MLK3 is a potent activator of the c-Jun N-terminal kinase (JNK) MAP kinases by directly phosphorylating and activating the JNK kinases, MKK4 and 7 [9,10]. MLK3 may also activate the p38 MAP kinases through direct phosphorylation of MKK3 and 6, which are the primary p38 MAP kinase activators [10].…”