title: Dimerization via tandem leucine zippers is essential for the activation of the mitogen-activated protein kinase kinase kinase, MLK-3.
abstract: Mixed lineage kinase-3 (MLK-3) is a mitogen-activated kinase kinase kinase that mediates stress-activating protein kinase (SAPK)/c-Jun NH2-terminal kinase activation. MLK-3 and other MLK family kinases are characterized by the presence of multiple protein-protein interaction domains including a tandem leucine/isoleucine zipper (LZs) motif. Leucine zippers are known to mediate protein dimerization raising the possibility that the tandem leucine/isoleucine zippers may function as a dimerization motif of MLK-3. Using both co-immunoprecipitation and nonreducing SDS-polyacrylamide gel electrophoresis, we demonstrated that MLK-3 forms disulfide bridged homo-dimers and that the LZs motif is sufficient for MLK-3 homodimerization. We next asked whether MLK-3 utilizes a dimerization-based activation mechanism analogous to that of receptor tyrosine kinases. We found that dimerization via the LZs motif is a prerequisite for MLK-3 autophosphorylation. We then demonstrated that co-expression of Cdc42 lead to a substantial increase in MLK-3 dimerization, indicating that binding by this GTPase may induce MLK-3 dimerization. Moreover, the LZs minus form of MLK-3 failed to activate the downstream target SAPK, and expression of a MLK-3 LZs polypeptide was found to block SAPK activation by wild type MLK-3. Taken together, these findings indicate that dimerization plays a pivotal role in MLK-3 activation.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: I W
authors.initials: IW
authors.lastname: Leung
authors.forename: N
authors.initials: N
authors.lastname: Lassam
mesh_terms: D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D017931:DNA Primers; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D016350:Leucine Zippers; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
chemical_list: D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004220:Disulfides; D048031:JNK Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11