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. 2014 Dec;13(12):3332-42.
doi: 10.1074/mcp.M114.041087. Epub 2014 Aug 25.

Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor

Sam Lievens  1 , Sarah Gerlo  1 , Irma Lemmens  1 , Dries J H De Clercq  2 , Martijn D P Risseeuw  2 , Nele Vanderroost  1 , Anne-Sophie De Smet  1 , Elien Ruyssinck  1 , Eric Chevet  3 , Serge Van Calenbergh  2 , Jan Tavernier  4
Affiliations

Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor

Sam Lievens et al. Mol Cell Proteomics. 2014 Dec.

Abstract

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges.

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Figures

Fig. 1.
Fig. 1.
KISS concept. A bait protein (X) is fused to a kinase-containing portion of TYK2 and a prey (Y) is coupled to a gp130 cytokine receptor fragment. When bait and prey interact, TYK2 phosphorylates STAT3 docking sites on the prey chimera (P), which ultimately leads to activation of a reporter gene (see text for more details).
Fig. 2.
Fig. 2.
Benchmarking KISS against other binary PPI methods. A, The PRS and RRS sets were tested in two complementary configurations with TYK2 fused either N- (configuration 1) or C-terminal (configuration 2) to the bait protein X; the prey fusions always contained the gp130 moiety fused N-terminally to the prey protein. B, KISS assay sensitivity (percentage of PRS positive; yellow bars) and assay specificity (percentage of RRS positive; blue bars) compared with that of orthogonal assays. C, Overview of the individual protein pairs of the PRS (top panel, yellow squares) and the RRS (bottom panel, blue squares) detected by KISS and the orthogonal assays. In B and C data for LUMIER, MAPPIT, Y2H, PCA, and wNAPPA have been extracted from Braun et al. (17). For details on thresholds used in the analysis of the KISS data, see Experimental Procedures.
Fig. 3.
Fig. 3.
In situ monitoring of PPIs and their modulation in response to external stimuli or a changing physiological context. A, Bait and prey fusion proteins can be either soluble or transmembrane proteins. The combinations shown in the cartoon correspond to the cases illustrated in the text. B, Analysis of interactions between HIV-1 RT p51 and p66 subunits. Luciferase data is normalized for control prey (unfused gp130). For raw luciferase data and Western blot controls see supplemental Fig. S2A. C, D, Dose-dependent KISS induction in cells co-expressing somatostatin receptor 2 (SSTR2; C) or angiotensin receptor 1 (AGTR1; D) bait and beta arrestin 2 (ARRB2) prey and treated with increasing concentrations of somatostatin (SST-14) or angiotensin II (AngII), respectively. E, Subcellular localization of ERN1 bait and prey fusion proteins. The ER-resident protein calnexin is used as an ER marker. ERN1 bait and prey localize to the ER (upper panel), and cluster in that compartment upon tunicamycin treatment (1 μg/ml; lower panel). Scale bars indicate 20 μm. F, Dose-dependent detection of ERN1 oligomerization upon treatment with tunicamycin. G, Analysis of ERN1 mutants in the presence or absence of tunicamycin. Cells expressing ERN1 wild type or point mutant (K599 or D123) baits combined with ERN1 or empty control prey and the luciferase reporter plasmid were treated with tunicamycin (1 μg/ml) or vehicle (DMSO). H, Inhibition of tunicamycin-induced ERN1 clustering by overexpression of BiP. Cells co-expressing ERN1 bait and prey together with BiP or actin were treated with tunicamycin (1 μg/ml) or vehicle (DMSO). Expression controls for Fig. 3F, 3G, and 3H are shown in supplemental Fig. S4C, S4D, and S4E, respectively.
Fig. 4.
Fig. 4.
Analysis of pharmacological interference with PPIs. A, B, Activity profile of the PPI inhibitors Nutlin-3 (A) and ABT-737 (B). Nutlin-3 and ABT-737 selectively disrupt the interaction between p53 and MDM2 (IC50 = 0.57 μm) or BAD and BCL2 (IC50 = 0.14 μm), respectively. MDM4 prey or unfused gp130 (ctrl) were used as control preys. C, D, Dose-dependent inhibition of the GPCR-beta arrestin 2 interaction by GPCR antagonists in cells expressing somatostatin receptor 1-beta arrestin 2 or angiotensin receptor 1-beta arrestin combinations as in Fig. 3C and 3D, and stimulated with the appropriate ligand (SST-14 or AngII; 10 μm).
Fig. 5.
Fig. 5.
KISS three-hybrid analysis of interactions between small molecules and their protein targets. A, Schematic overview of the three-hybrid KISS setup. eDHFR (“D”) is tethered to a C-terminal kinase-domain-containing TYK2 fragment and co-expressed with a prey protein (“Y”) fused to a gp130 cytokine receptor fragment that contains STAT3 docking sites (black dots). When cells are treated with a fusion compound in which a small molecule of interest (asterisk) is linked to methotrexate (MTX) through a polyethyleneglycol linker, MTX binds to eDHFR with very high affinity, resulting in the small molecule being presented as bait. Interaction between the small molecule bait and the protein prey brings TYK2 in the proximity of the gp130 receptor tail, allowing the kinase to phosphorylate the STAT docking sites on the prey chimera (“P”). This leads to the recruitment of STAT3 molecules, which are in turn phosphorylated by the kinase, resulting in their activation. Activated STATs migrate to the nucleus where dimers of this transcription factor activate transcription of a reporter gene. B, KISS analysis of the interaction between the small molecules FK506 and simvastatin with their respective protein targets FKBP12 and HMGCR, respectively. A luciferase signal was generated specifically when eDHFR bait is co-expressed with FKBP12 or HMGCR prey fusions and treated with the appropriate MFC (MTX-FK506 and MTX-simvastatin, respectively; 5 μm). No signal was observed in unstimulated cells or in the presence of unfused gp130 as control prey. Control of bait and prey expression by Western blot is presented in Supplemental Fig. S5. C, D, Dose-dependent detection of the interaction between MTX-derivatized FK506 (C) and simvastatin (D) and their respective targets, FKBP12 and HMGCR. Structure of the MTX fusion compounds are shown below the corresponding curves.
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