CHARACTERIZATION OF DUAL LEUCINE ZIPPER-BEARING KINASE, A MIXED LINEAGE KINASE PRESENT IN SYNAPTIC TERMINALS WHOSE PHOSPHORYLATION STATE ISREGULATED BY MEMBRANE DEPOLARIZATION VIA CALCINEURIN

The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protei n kinases, was examined in the nervous system, DLK transcript expressi on in the nervous system was predominantly neuronal, DLK protein was p resent in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had di ffering characteristics. Cytosolic DLK existed in both a phosphorylate d and dephosphorylated state; DLK associated with plasma membrane exis ted in the dephosphorylated state only, On nonreducing SDS-polyacrylam ide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while memb rane associated DLK migrated with an apparent M(r) greater than or equ al to 260,000, Similarly, DLK transiently expressed in COS 7 cells aut ophosphorylated in vivo and migrated at approximately 260 kDa when sep arated by nonreducing SDS polyacrylamide gel electrophoresis, In cotra nsfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-Delta 520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conc lusion that DLK formed covalently associated homodimers in overexpress ing COS 7 cells, In aggregating neuronal-glial cultures, depolarizatio n of plasma membrane lead to dephosphorylation of DLK, Treatment of ag gregates with 5 nM or 200 nM okadaic acid lead to a shift in electroph oretic mobility consistent with phosphorylation of DLK, Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulin-depende nt protein phosphatase 2B (calcineurin), had no effect on DLK phosphor ylation under basal conditions, However, cyclosporin A completely inhi bited DLK dephosphorylation upon membrane depolarization.