KinCon: Cell‐based recording of full‐length kinase conformations

Enzler, Florian; Tschaikner, Philipp; Schneider, Rainer; Stefan, Eduard

doi:10.1002/iub.2241

Cited by 13 publications

(24 citation statements)

References 56 publications

(88 reference statements)

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“…However, it is clear that evaluating the mechanistic outputs of all human pseudokinases, and establishing their biological functions and respective cellular niches, remains a major research goal. For example, although approximately half of human pseudokinases are still able to bind to nucleotides, the functional consequences of this event remain obscure in nearly all cases, perhaps due to a current lack of cellular reporters designed to report pseudokinase conformations . Ever‐closer collaboration between specialists in various fields fully supports the notion that molecular dissection of pseudokinase‐based signalling mechanisms will be enhanced if the decades of combined experience focused on analysing (canonical) protein kinases can be concentrated and then refocused.…”

Section: Introductionmentioning

confidence: 98%

Cataloguing the dead: breathing new life into pseudokinase research

et al. 2020

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Pseudoenzymes are present within many, but not all, known enzyme families and lack one or more conserved canonical amino acids that help define their catalytically active counterparts. Recent findings in the pseudokinase field confirm that evolutionary repurposing of the structurally defined bilobal protein kinase fold permits distinct biological functions to emerge, many of which rely on conformational switching, as opposed to canonical catalysis. In this analysis, we evaluate progress in evaluating several members of the 'dark' pseudokinome that are pertinent to help drive this expanding field. Initially, we discuss how adaptions in erythropoietin-producing hepatocellular carcinoma (Eph) receptor tyrosine kinase domains resulted in two vertebrate pseudokinases, EphA10 and EphB6, in which co-evolving sequences generate new motifs that are likely to be important for both nucleotide binding and catalysis-independent signalling. Secondly, we discuss how conformationally flexible Tribbles pseudokinases, which have radiated in the complex vertebrates, control fundamental aspects of cell signalling that may be targetable with covalent small molecules. Finally, we show how species-level adaptions in the duplicated canonical kinase protein serine kinase histone (PSKH)1 sequence have led to the appearance of the pseudokinase PSKH2, whose physiological role remains mysterious. In conclusion, we show how the patterns we discover are selectively conserved within specific pseudokinases, and that when they are modelled alongside closely related canonical kinases, many are found to be located in functionally important regions of the conserved kinase fold. Interrogation of these patterns will be useful for future evaluation of these, and other, members of the unstudied human kinome.

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Section: Introductionmentioning

confidence: 98%

Cataloguing the dead: breathing new life into pseudokinase research

et al. 2020

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“…We recently demonstrated that α-C-OUT BRAF inhibitors (BRAFi) show an allosteric effect on mutated and full-length BRAF-V600E conformations. We showed that the recorded conformational change reflects enzyme activities with the BRAF-V600E KinCon reporter ( 13 , 42 ). These KinCon reporter activity profiles directly correlate with activity measurements of ERK kinase activation ( 13 ).…”

Section: Resultsmentioning

confidence: 99%

“…Adaptable biosensor technologies for intramolecular protein dynamics are still missing. We engineered genetically encoded biosensors to quantify autoinhibitory conformation changes of kinases and other signaling proteins involved in oncogenic signaling ( 13 , 42 ).…”

Section: Resultsmentioning

confidence: 99%

“…FRET and BRET reporters have been described for measuring RAF kinase protein–protein interactions (PPI) and dynamics ( 14 , 46 , 47 ). We set out to apply a protein-fragment complementation assay (PCA)–based kinase conformation (KinCon) reporter platform to systematically record the impact of combinations of mutations and approved kinase inhibitors or lead molecules on enzyme activity conformations ( 13 , 42 ). We integrated the most frequent BRAF cancer patient mutations into the KinCon platform for tailored drug discovery efforts to be performed directly in the target cell.…”

Section: Resultsmentioning

confidence: 99%

“…1 A ) are members of different kinase families. The closed conformation of autoinhibitory proteins occasionally requires that the original protein termini are brought into vicinity ( 9 , 13 , 36 – 38 , 42 , 57 ). We utilized this fact and fused this handpicked collection of kinases N-terminally with fragment 1 (F[1]-) and C-terminally with fragment 2 (-F[2]) of the R luc-based PCA as illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies

Mayrhofer

Enzler

Feichtner

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non–small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein–protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

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