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

Abstract

e 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 ␣-actinin-1 and 14-3-3-␤ are novel DAPK2 binding partners. The interaction of DAPK2 with ␣-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-␤ 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. D eath-associated protein kinases (DAPKs) are a family of five Ser/Thr kinases that share a high degree of sequence homology in their catalytic domains but differ significantly in their extracatalytic domains. The best-studied member of the DAPK family is DAPK1, a regulator of programmed cell death (1, 2), autophagy (3), and motility (4).DAPK2 exhibits a kinase domain with 80% homology to the one of DAPK1 and also contains a calmodulin (CaM)-binding site (5, 6) but uniquely features a C-terminal homodimerization domain (6) while lacking the diverse protein-protein interaction domains that DAPK1 possesses (5, 6). The kinase is kept in an inactive conformation by a double-locking mechanism, which requires dephosphorylation of an autophosphorylated residue (Ser318) within the CaM-binding domain by a yet-to-be identified phosphatase in order to allow for CaM binding and homodimerization, both of which enhance kinase activity (7,8). Also the phosphorylation of Ser299 by cyclic GMP (cGMP)-dependent protein kinase I (9) and the interaction with 14-3-3-(10) regulate DAPK2 kinase activity. To date, the only known DAPK2 substrates are DAPK2 itself, regulatory light chain of myosin II (RLC), and mammalian target of rapamycin complex 1 (mTORC1) (5, 6, 11).DAPK2 was shown to mediate programmed cell death. Overexpression of DAPK2 results in morphological changes reminiscent of apoptosis in adherent cells, such as membrane blebbing and condensation of nuclei (5, 6), and in reduced viability and survival in suspension cells (12,13). In line with these findings, restoration of DAPK2 activity through fusion of a consti...

Death‐associated protein kinase (DAPK) family modulators: Current and future therapeutic outcomes

“…In addition to regulation by Ca +2 /CaM regulatory domain, DAPK2 is regulated by binding to 14‐3‐3 proteins . The 14‐3‐3 proteins family is a family of regulatory proteins that bind phosphoserine‐containing proteins regulating their biological functions and thus modulating diverse biological activities .…”

“…Binding of 14‐3‐3 proteins to DAPK2 depends on phosphorylation of Thr369 residue on the C‐terminal tail of DAPK2 by protein kinase B (Akt). However, blocking the activity of Akt did not completely abolish this binding due to the ability of other kinases such as RSK and protein kinase A (PKA) to phosphorylate serine and arginine residues within the tail of DAPK2 …”

GOLPH3 Promotes Cancer Growth by Interacting With STIP1 and Regulating Telomerase Activity in Pancreatic Ductal Adenocarcinoma

“…To gain insights into the molecular mechanisms of GOLPH3's biological functions, we identified potential GOLPH3 interaction partners using a high-throughput bimolecular fluorescence complementation (BiFC) assay, a method that can visualize protein interactions in living cells ( 21 ). This method is based on the discovery that two non-fluorescent fragments of a fluorescent protein can form a fluorescent entity when in close proximity ( 22 ). The BiFC strategy has been utilized for a variety of applications, including the visualization of protein interactions ( 21 ), determination of subcellular localizations ( 23 ), and investigation of biological functions of PPIs ( 24 ).…”

miR-1285-3p Controls Colorectal Cancer Proliferation and Escape from Apoptosis through DAPK2

“…The N-terminal region of DAPK2, spanning the kinase domain and the CaM regulatory domain, shows a high degree of sequence similarity with DAPK1, the founding member of this protein family, whereas the C-terminal tail is required for self-dimerization. Only a few interactors of DAPK2 have been identified so far [32][33][34]. Accordingly, just a limited number of DAPK2 substrates is currently known: myosin II regulatory light chain, the mTOR binding partner raptor [35], the autophagy receptor protein p62/SQSTM [36] and the core autophagic machinery protein Beclin-1 [37].…”