Dataset Information

Phosphorylation-Assisted Luciferase Complementation Assay Designed to Monitor Kinase Activity and Kinase-Domain-Mediated Protein-Protein Binding.

ABSTRACT: Protein kinases are key regulators of cell signaling and have been important therapeutic targets for three decades. ATP-competitive drugs directly inhibit the activity of kinases but these enzymes work as part of complex protein networks in which protein-protein interactions (often referred to as kinase docking) may govern a more complex activation pattern. Kinase docking is indispensable for many signaling disease-relevant Ser/Thr kinases and it is mediated by a dedicated surface groove on the kinase domain which is distinct from the substrate-binding pocket. Thus, interfering with kinase docking provides an alternative strategy to control kinases. We describe activity sensors developed for p90 ribosomal S6 kinase (RSK) and mitogen-activated protein kinases (MAPKs: ERK, p38, and JNK) whose substrate phosphorylation is known to depend on kinase-docking-groove-mediated protein-protein binding. The in vitro assays were based on fragment complementation of the NanoBit luciferase, which is facilitated upon substrate motif phosphorylation. The new phosphorylation-assisted luciferase complementation (PhALC) sensors are highly selective and the PhALC assay is a useful tool for the quantitative analysis of kinase activity or kinase docking, and even for high-throughput screening of academic compound collections.

SUBMITTER: Poti AL

PROVIDER: S-EPMC10573712 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Phosphorylation-Assisted Luciferase Complementation Assay Designed to Monitor Kinase Activity and Kinase-Domain-Mediated Protein-Protein Binding.

Póti Ádám L ÁL Dénes Laura L Papp Kinga K Bató Csaba C Bánóczi Zoltán Z Reményi Attila A Alexa Anita A

International journal of molecular sciences 20231003 19

Protein kinases are key regulators of cell signaling and have been important therapeutic targets for three decades. ATP-competitive drugs directly inhibit the activity of kinases but these enzymes work as part of complex protein networks in which protein-protein interactions (often referred to as kinase docking) may govern a more complex activation pattern. Kinase docking is indispensable for many signaling disease-relevant Ser/Thr kinases and it is mediated by a dedicated surface groove on the ...[more]

PMID: 37834301

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Project description:A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Project description:The regulation of organismic lipid storage amounts is a central goal of all organisms. Proteins associated with the dedicated lipid storage organelles, the so-called lipid droplets (LDs), are instrumental to this regulation. LD proteomes of diverse organisms have been characterized by mass spectrometry. Yet, the functional characterization of most of these proteins is still lagging behind. Moreover, knowledge of interactions among LD-associated proteins is sparse and mostly limited to proteins involved in mammalian lipolysis regulation. This process crucially depends on an orchestrated set of protein-protein interactions which limit the accessibility, and thus remobilization, of the lipid stores. Thus, a better knowledge of the LD-associated protein interactome likely reveals entry points to deeper understand lipid storage regulation on a mechanistic level. Here, we utilize a split-luciferase based protein-protein interaction assay to identify interactions among 46 LD-associated proteins and protein variants of which 40 are Drosophila and 6 mouse proteins. In 1531 complementation tests, we assayed for interactions among 487 luciferase fragment fusion protein pairs (covered by 830 different construct combinations) of which 85 (17.45 %) resulted in a significant luciferase complementation. These results suggest, that a prominent fraction of the LD-associated proteome participates in protein-protein interactions, and provide a basis for further functional studies. Besides confirming previously described interactions, we identified several new protein interaction pairs. Of these, we characterize the following three in greater detail: (i) Jabba and CG9186, (ii) Jabba and Histones and (iii) Ubiquitin and CG9186. The interaction among Jabba and Histones is of physiological significance and we provide data suggesting that Jabba alone is sufficient to recruit Histones to LDs. Further, we identified a positively charged amino acid stretch of 16 amino acids within the Jabba protein which is necessary for the Jabba-Histone interaction. CG9186 is an annotated esterase which is involved in lipid storage amount regulation and positioning of LDs. We present data supporting the ubiquitination of at least two lysine residues of CG9186 and demonstrate a role of CG9186 ubiquitination in the induction of LD clusters linked to the overexpression of CG9186.

Dataset Information

Phosphorylation-Assisted Luciferase Complementation Assay Designed to Monitor Kinase Activity and Kinase-Domain-Mediated Protein-Protein Binding.

Publications

Phosphorylation-Assisted Luciferase Complementation Assay Designed to Monitor Kinase Activity and Kinase-Domain-Mediated Protein-Protein Binding.

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