Dimerization via tandem leucine zippers is essential for the activation ofthe mitogen-activated protein kinase kinase kinase, MLK-3

Mixed lineage kinase-3 (MLK-3) is a mitogen-activated kinase kinase kinase that mediates stress-activating protein kinase (SAPK)/c-Jun NH2-terminal ki nase activation. MLK-3 and other MLK family kinases are characterized by th e presence of multiple protein-protein interaction domains including a tand em leucine/isoleucine zipper (LZs) motif, Leucine zippers are known to medi ate protein dimerization raising the possibility that the tandem leucine/is oleucine zippers may function as a dimerization motif of MLK-3. Using both co-immunoprecipitation and nonreducing SDS-polyacrylamide gel electrophores is, we demonstrated that MLK-3 forms disulfide bridged homo-dimers and that the LZs motif is sufficient for MLK-3 homodimerization. We next asked whet her MLK-3 utilizes a dimerization-based activation mechanism analogous to t hat of receptor tyrosine kinases. We found that dimerization via the LZs mo tif is a prerequisite for MLK-3 autophosphorylation. We then demonstrated t hat co-expression of Cdc42 lead to a substantial increase in MLK-3 dimeriza tion, indicating that binding by this GTPase may induce MLK-3 dimerization. Moreover, the LZs minus form of MLK-3 failed to activate the downstream ta rget SAPK, and expression of a MLK-3 LZs polypeptide was found to block SAP K activation by wild type MLK-3. Taken together, these findings indicate th at dimerization plays a pivotal role in MLK-3 activation.