Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Geering, Barbara; Zokouri, Zina; Hürlemann, Samuel; Gerrits, Bertran; Ausländer, David; Britschgi, Adrian; Tschan, Mario; Simon, Hans-Uwe; Fussenegger, Martin (2015). Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation. Molecular and cellular biology, 36(1), pp. 132-143. American Society for Microbiology 10.1128/MCB.00515-15

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Pharmacology
04 Faculty of Medicine > Service Sector > Institute of Pathology > Tumour Pathology
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Med. Onkologie / Hämatologie (Erw.)
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Med. Onkologie / Hämatologie (Erw.)

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Britschgi, Adrian; Tschan, Mario and Simon, Hans-Uwe

Subjects:

600 Technology > 610 Medicine & health

ISSN:

0270-7306

Publisher:

American Society for Microbiology

Language:

English

Submitter:

Doris Haefelin

Date Deposited:

21 Jan 2016 16:32

Last Modified:

06 Jul 2016 20:55

Publisher DOI:

10.1128/MCB.00515-15

PubMed ID:

26483415

BORIS DOI:

10.7892/boris.74618

URI:

https://boris.unibe.ch/id/eprint/74618

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