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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with
Barbara Geering1, Zina Zokouri2, Samuel Hürlemann2
1Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland barbara.geering@bsse.ethz.ch.
Abstract:
Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.
Insights
Death-associated protein kinase 2 (DAPK2) has 180 potential binding partners identified, revealing novel interactions with α-actinin-1 and 14-3-3-β. These interactions influence DAPK2
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Death-associated protein kinase 2 (DAPK2) is a serine/threonine kinase with tumor-suppressive functions.
- DAPK2 regulates critical cellular processes including programmed cell death, autophagy, and motility.
- The molecular mechanisms underlying DAPK2's functions are poorly understood due to limited knowledge of its binding partners.
Purpose of the Study:
- To identify novel protein-protein interactions of DAPK2.
- To elucidate the molecular mechanisms governing DAPK2's biological functions.
- To investigate the role of subcellular localization in DAPK2 effector functions.
Main Methods:
- Affinity purification coupled with mass spectrometry (AP-MS) was employed to identify DAPK2 interaction partners.
- Bimolecular fluorescence complementation (BiFC) assays were used to visualize and confirm protein interactions in living cells.
- Subcellular localization of DAPK2 interactions was analyzed.
Main Results:
- 180 potential DAPK2 interaction partners were identified, including 12 known binding proteins.
- α-actinin-1 and 14-3-3-β were confirmed as novel DAPK2 binding partners.
- DAPK2 interaction with α-actinin-1 at the plasma membrane induced membrane blebbing and reduced motility.
- DAPK2 interaction with 14-3-3-β in the cytoplasm had no significant impact on cellular processes.
Conclusions:
- DAPK2's effector functions are modulated by its subcellular localization.
- Combining AP-MS and BiFC is effective for identifying and characterizing novel protein-protein interactions.
- This study expands the understanding of DAPK2 interactome and its functional regulation.
