Characterization of Dual Leucine Zipper-bearing Kinase, a Mixed Lineage Kinase Present in Synaptic Terminals Whose Phosphorylation State Is Regulated by Membrane Depolarization via Calcineurin

Abstract

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.

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.…”

“…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].…”

“…In aggregating neuronal-glial cultures, the inhibition by cyclosporin A of the calcium/calmodulin-dependent protein phosphatase calcineurin prevented a membrane depolarisation-induced shift in the electrophoretic mobility of DLK [19], suggesting that the phosphorylation state of DLK may activity. The plasmid 5×Gal4E1BLuc (2 μg) was cotransfected with an expression vector for GAL4-CBP (2 μg) and with 2 μg of Bluescript (minus sign) or 2 μg of the expression vector for wildtype DLK (DLK).…”

“…The plasmids p4xSomCRELuc [2], 5xGal4E1BLuc [3], and the expression vectors for DLK and DLKmt [19], calcineurin subunits A and B [20], GAL4-CBP, containing the C-terminal amino acids of CBP from 1,678 to 2,441 (kind gift of R. H. Goodman, Oregon Health and Sciences University, Portland, OR, USA) [8] have been described before. The expression vector for glutathione S-transferase (GST)-c-Jun (amino acids 1-135) was kindly provided by M. Kracht, Medical School Hanover, Hanover, Germany [21].…”

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

“…The MLK family includes MLK1-4, the dual leucine zipper-bearing kinase (DLK), and the leucine zipper kinase (LZK) [1,2]. MLK proteins each contain highly conserved structural motifs that are important for protein interactions and signal transduction including a Src homology 3 (SH3) domain, a catalytic domain, two leucine zipper (LeuZip) motifs, and a Cdc42/Rac interactive binding (CRIB) motif [1].…”