Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.
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.
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland barbara.geering@bsse.ethz.ch.
Barbara
http://orcid.org/0000-0001-7207-5819
B
Geering
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Zina
Z
Zokouri
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Samuel
S
Hürlemann
Functional Genomics Center Zurich, ETH Zurich, Zurich, Switzerland.
Bertran
B
Gerrits
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
David
D
Ausländer
Institute of Pathology, University of Bern, Bern, Switzerland.
Adrian
A
Britschgi
Institute of Pathology, University of Bern, Bern, Switzerland.
Mario P
MP
Tschan
Institute of Pharmacology, University of Bern, Bern, Switzerland.
Hans-Uwe
HU
Simon
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Faculty of Science, University of Basel, Basel, Switzerland.
Martin
M
Fussenegger
D017209:Apoptosis / Q000502:physiology*; D001343:Autophagy / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D064550:Death-Associated Protein Kinases / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D040901:Proteomics / Q000379:methods
C579104:DAPK2 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases