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

doi:10.1074/jbc.274.15.10195

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

From the ^¶Department of Veterans Affairs, Ann Arbor, Michigan 48105, the ^‡Division of Nephrology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, 48109-0676, the ^§Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, and the Department of Veterans Affairs, Pittsburgh, Pennsylvania 15213

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 NH₂-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.

Footnotes

↵* This work was supported in part by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to L. B. H., and M. M.), and by the National Institutes of Health (to L. B. H., and M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: University of Michigan Medical School, 1560 Medical Science Research Bldg. II, Ann Arbor, MI 48109-0676. Tel.: 734-764-3157; Fax: 734-763-0982; E-mail:lholzman{at}umich.edu.
Abbreviations:

MAPK

mitogen-activated protein kinase

MAPKK (also MEK and MKK)

mitogen-activated protein kinase kinase

MAPKKK

mitogen-activated protein kinase kinase kinase

SAPK

stress-activated protein kinase

JNK

c-Jun NH₂-terminal kinase

MLK

mixed lineage kinsae

DLK

dual leucine zipper-bearing kinase

PBS

phosphate-buffered saline

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis
- Received September 15, 1998.
- Revision received January 13, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.