KISS: A Mammalian Two-Hybrid Method for In Situ Analysis of Protein–Protein Interactions

Abstract

KISS (KInase Substrate Sensor) is a recently developed two-hybrid technology that allows in situ analysis of protein-protein interactions in intact mammalian cells. In this method, which is derived from MAPPIT (mammalian protein-protein interaction trap), the bait protein is coupled to the kinase domain of TYK2, while the prey protein is fused to a fragment of the gp130 cytokine receptor chain. Bait and prey interaction leads to phosphorylation of the gp130 anchor by TYK2, followed by recruitment and activation of STAT3, resulting in transcription of a STAT3-dependent reporter system. This approach enables the identification of interactions between proteins, including transmembrane and cytosolic proteins, and their modulation in response to physiological or pharmacological challenges. Here, we describe a detailed step-by-step protocol for the detection of an interaction between two proteins of interest using KISS.

An Unexpected Encounter: Respiratory Syncytial Virus Nonstructural Protein 1 Interacts with Mediator Subunit MED25

“…As such, we wanted to obtain a more comprehensive overview of candidate NS1 interaction partners and intended to focus on overlapping hits. MAPPIT and KISS are two-hybrid approaches that are based on the complementation of cytokine signal transduction ( 26 – 29 ). The prey collection that was used in the MAPPIT and KISS screens consists of 14,817 proteins, selected from the human ORFeome collection version 8.1 and the ORFeome collaboration (OC) collection ( 35 , 36 ).…”

“…For the construction of the KISS bait vector, the coding sequence of RSV A2 NS1 was fused to the N terminus of a fragment of human TYK2 (amino acids 589 to 1187) and inserted into the pMet7 expression vector. The construction of the preys, the primary KISS screen, and retest screens was performed as described previously ( 27 ).…”

“…For both MAPPIT and KISS, the simple reporter gene readout enables high-throughput screening for covalent, transient, and indirect interactions in intact mammalian cells. It is important to note that in MAPPIT, the bait is anchored to the plasma membrane, which may not be the native subcellular localization of the bait, whereas in KISS, the bait is expressed as a cytosolic fusion protein ( 27 ).…”

“…We also set out to investigate the NS1 interactome with two binary methods, MAPPIT and KISS, which are based on cytokine signal transduction ( 26 , 27 ). Briefly, MAPPIT uses an engineered chimeric receptor that consists of the extracellular part of the erythropoietin (EPO) and the cytosolic part of the leptin receptor ( 28 ).…”

TYK2: An Upstream Kinase of STATs in Cancer

“…The mapped and predicted PPIs of TYK2 based on proteomics [138,139] and next generation sequencing (NGS) are accessible at various open-source databases (for a review, see [140]). The TYK2 kinase domain and a STAT3-based reporter system were used to establish the first mammalian two hybrid kinase substrate sensor (KISS) screening platform [141,142]. These databases and the screening approaches should be systematically exploited to further define and fine tune the TYK2 interactome in health and disease.…”