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.
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 Mr >/= 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-Delta520) 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/calmodulin-dependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.
Mata
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Department of Neurology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania 15261, USA.
Merritt
S E
SE
Fan
G
G
Yu
G G
GG
Holzman
L B
LB
D000818:Animals; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology*; D002384:Catalysis; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism; D016350:Leucine Zippers; D020930:MAP Kinase Kinase Kinases; D008564:Membrane Potentials / Q000187:drug effects*; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology*; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D016548:Prosencephalon / Q000166:cytology / Q000201:enzymology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats
D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D004220:Disulfides; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C090757:mitogen-activated protein kinase kinase kinase 12; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases