Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

Abstract

An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit Cbeta1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of Cbeta1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes.

Fine-Tuning Multiprotein Complexes Using Small Molecules

“…Other HTS methods have also been specially designed to favor the identification of PPI inhibitors or activators. For example, chemoproteomic profiling allows measurements of drug-induced changes in protein complex formation (93, 94). Another promising approach is “grey-box” screening, in which multi-protein complexes are re-constituted in vitro and then an HTS method is used to find molecules that impact the biochemical properties of the complex.…”

“…Other HTS methods have also been specially designed to favor the identification of PPI inhibitors or activators. For example, chemoproteomic profiling allows measurements of drug-induced changes in protein complex formation. , Another promising approach is “grey-box” screening, in which multiprotein complexes are reconstituted in vitro , and then an HTS method is used to find molecules that impact the biochemical properties of the complex. For example, in a screen for inhibitors of the p21-activated kinase (Pak1), Deacon and colleagues chose to use the full-length protein, including its non-catalytic domain, in complex with an activator protein, Cdc42, and a substrate, maltose binding protein .…”

PRKACB is downregulated in non-small cell lung cancer and exogenous PRKACB inhibits proliferation and invasion of LTEP-A2 cells

“…A recent study discovered a novel interaction between PRKACB, the cell cycle and CARP-1; this was confirmed by GST pull-down experiments in brain tissue (16). A study has also demonstrated that PRKACB interacts with p75NTR, which phosphorylates p75NTR at Ser304 (14).…”

“…PRKACB has been considered to prevent the overgrowth of cells by inducing cell apoptosis (15,16). Therefore, apoptosis was examined following PRKACB transfection using Annexin V-PE/7-AAD assay and flow cytometry.…”

“…PRKACB was identified as a candidate gene that is directly or indirectly involved in apoptosis in human mantle cell lymphoma (MCL) tumors (15). In addition, a novel interaction between PRKACB, the cell cycle and apoptosis regulatory protein-1 (CARP-1) was identified and confirmed by glutathione-S-transferase (GST) pull-down experiments in brain tissue (16). However, limited information is known with regard to its expression and role in human NSCLC.…”