title: Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor.
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.
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium;
authors.forename: Sam
authors.initials: S
authors.lastname: Lievens
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium;
authors.forename: Sarah
authors.initials: S
authors.lastname: Gerlo
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium;
authors.forename: Irma
authors.initials: I
authors.lastname: Lemmens
authors.affiliation: ¶Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium;
authors.forename: Dries J H
authors.initials: DJ
authors.lastname: De Clercq
authors.affiliation: ¶Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium;
authors.forename: Martijn D P
authors.initials: MD
authors.lastname: Risseeuw
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium;
authors.forename: Nele
authors.initials: N
authors.lastname: Vanderroost
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium;
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: De Smet
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium;
authors.forename: Elien
authors.initials: E
authors.lastname: Ruyssinck
authors.affiliation: ‖French National Institute for Health and Medical Research (INSERM) U1053, University of Bordeaux Segalen, 146 Rue Leo Saignat, 33000 Bordeaux, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Chevet
authors.affiliation: ¶Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium;
authors.forename: Serge
authors.initials: S
authors.lastname: Van Calenbergh
authors.affiliation: From the ‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium ; §Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium; jan.tavernier@vib-ugent.be.
authors.forename: Jan
authors.initials: J
authors.lastname: Tavernier
mesh_terms: D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D019985:Benchmarking; D015374:Biosensing Techniques / Q000379:methods*; D059865:Endoplasmic Reticulum Stress / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism; D017481:Receptors, Somatostatin / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D053634:TYK2 Kinase / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D000071557:beta-Arrestins
chemical_list: C544065:AGTR1 protein, human; D019390:Arrestins; D044140:Receptor, Angiotensin, Type 1; D017481:Receptors, Somatostatin; C574734:SSTR2 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D000071557:beta-Arrestins; D053634:TYK2 Kinase; C070139:TYK2 protein, human; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases