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Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain
1Cell and Molecular Biology Program, Michigan State University, East Lansing, Michigan 48824, USA.
Abstract:
Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.
Insights
Mixed lineage kinase 3 (MLK3) is negatively regulated by its own Src homology 3 (SH3) domain. Disrupting the SH3 domain increases MLK3
Area of Science:
- Molecular Biology
- Protein Kinase Regulation
- Signal Transduction
Background:
- Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase involved in activating the c-Jun NH(2)-terminal kinase and NF-kappa B pathways.
- MLK3 possesses a Src homology 3 (SH3) domain N-terminal to its catalytic domain, crucial for protein-protein interactions.
Purpose of the Study:
- To investigate the regulatory role of the MLK3 SH3 domain in kinase activity.
- To identify the interaction site and mechanism of SH3 domain-mediated regulation of MLK3.
Main Methods:
- Site-directed mutagenesis was used to create an SH3-defective MLK3 mutant.
- In vitro kinase assays were performed to compare the activity of wild-type and mutant MLK3.
- Protein interaction mapping identified the specific binding region between the SH3 domain and MLK3.
Main Results:
- The SH3-defective MLK3 mutant displayed significantly higher catalytic activity than wild-type MLK3, indicating negative regulation.
- The MLK3 SH3 domain was found to interact with the full-length MLK3 protein.
- Interaction mapping revealed a unique binding site involving a single proline residue, mutation of which abrogated binding and increased kinase activity.
Conclusions:
- MLK3 is autoinhibited by its SH3 domain through a novel mechanism involving a single proline residue.
- This SH3 domain-mediated autoinhibition is likely conserved in related kinases like MLK1 and MLK2.
- The findings provide the first example of SH3 domain-mediated autoinhibition in a serine/threonine kinase, offering insights into mixed lineage kinase regulation.