Discovering Protein-Protein Interactions within the Programmed Cell Death Network Using a Protein-Fragment Complementation Screen

Abstract

Apoptosis and autophagy are distinct biological processes, each driven by a different set of protein-protein interactions, with significant crosstalk via direct interactions among apoptotic and autophagic proteins. To measure the global profile of these interactions, we adapted the Gaussia luciferase protein-fragment complementation assay (GLuc PCA), which monitors binding between proteins fused to complementary fragments of a luciferase reporter. A library encompassing 63 apoptotic and autophagic proteins was constructed for the analysis of ∼3,600 protein-pair combinations. This generated a detailed landscape of the apoptotic and autophagic modules and points of interface between them, identifying 46 previously unknown interactions. One of these interactions, between DAPK2, a Ser/Thr kinase that promotes autophagy, and 14-3-3τ, was further investigated. We mapped the region responsible for 14-3-3τ binding and proved that this interaction inhibits DAPK2 dimerization and activity. This proof of concept underscores the power of the GLuc PCA platform for the discovery of biochemical pathways within the cell death network.

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

“…Expression of split YFP-DAPK2 and -CaM or -␣-actinin-1 greatly enhanced the number of cells with a blebbing phenotype compared to levels in control cells, with the majority of cells demonstrating a blebbing phenotype following ectopic expression of split YFP-␣-actinin-1 and -DAPK2. In contrast, similar numbers of cells with a blebbing phenotype were observed when the interaction of split YFP-DAPK2 with -14-3-3-␤ compared to that in control cells was analyzed, contrary to previous reports in cells overexpressing DAPK2 and 14-3-3-, which showed reduced levels of blebbing (10). Expression of a split YFP construct fused to an inactive DAPK2 mutant (YFP N -K52A and YFP C -K52A) resulted in only a few blebbing cells in combination with split YFP-DAPK2, -CaM and -14-3-3-␤, suggesting that DAPK2 activity was essential for the observed phenotype.…”

“…In addition, 14-3-3-␤ was chosen for further investigations because, as for ␣-actinin-1, the protein was identified with high prevalence by LC-MS/MS. Furthermore, 14-3-3-␤, which is a phospho-Ser/phospho-Thr binding protein with a vast variety of functions (34), was previously shown to interact with DAPK1 in hippocampal areas surviving a seizure (35), and 14-3-3-␤ and 14-3-3-have recently been demonstrated to interact with phosphorylated DAPK2 (10,29).…”

“…Also, 14-3-3-␤ interacted directly with DAPK2 and, interestingly, independent of any phosphorylated residue. As 14-3-3-␤ and 14-3-3-have been shown to interact with a Ser/Thrrich stretch at the very C terminus of DAPK2 only when these residues were phosphorylated (10,29), our data suggesting that binding of DAPK2 with 14-3-3-␤ may also occur through different molecular mechanisms may be reminiscent of the interaction of 14-3-3-␤ with DAPK1 (35). We furthermore demonstrate that the interaction of DAPK2 with ␣-actinin-1 and 14-3-3-␤ affects the subcellular localization of DAPK2.…”

DAPK2 Downregulation Associates With Attenuated Adipocyte Autophagic Clearance in Human Obesity

“…H: Bars represent quantifications of autophagic flux (i.e., LC3-II/actin accumulation) in the presence vs. absence of lysosome inhibitor as mean values 6 SEM. Asterisks indicate statistical significance for P values: *P < 0.05, **P < 0.01, or ***P < 0.001. the beclin interactome (50,51). Interestingly, DAPK2 silencing was also found to affect tumor necrosis factorrelated apoptosis-inducing ligand signaling and nuclear factor-kB activation (52), reinforcing connections to cell inflammation.…”

The programmed cell death GLuc PCA library – a powerful tool for pathway discovery and drug screening

“…The library is based on the Gaussia luciferase protein-fragment complementation assay (PCA) reporter, which enables quantitative measurement of protein-protein interactions in a reversible manner. 4 The library encompasses 63 proteins, including apoptosis proteins, autophagy proteins, death-associated proteins (DAPs), and additional cell death regulatory proteins.…”