Figures and data
Kinase regulation and KinCon reporter technology features.
A) Impact of indicated factors/features (e.g. Protein-protein interactions (PPIs), post-translational modifications (PTM), cis-regulatory elements (CRE)) on the switch-like behavior of kinases. B) Schematic representation of the KinCon reporter technology using the Renilla Luciferase (Rluc) protein-fragment complementation assay (PCA) as it works for kinases such as BRAF which contain autoinhibitory modules (AIM); RLuc fragments 1 and 2 are N and C terminally fused to the kinase of interest (with interjacent linker in red) and are labeled with F[1] and F[2]. PPIs, drug (candidate) or small molecule binding, mutations and/or PTMs may convert the KinCon reporter into different conformation states. Protein movements are quantified through measuring alterations of bioluminescence signals uponRLuc substrate addition. C) Shown is the workflow for the KinCon reporter construct engineering and analyses using KinCon technology. The kinase gene of interest is inserted into the multiple cloning site of a mammalian expression vector which is flanked by respective PCA fragments (-F[1]-, -F[2]) and separated with interjacent flexible linkers. Expression of the genetically encoded reporter in indicated multi-well formats allows to vary expression levels and define a coherent drug treatment plan. Moreover, it is possible to alter the kinase sequence (mutations) or to co-express or knock-down the respective endogenous kinase, interlinked kinases or proteinogenic regulators of the respective pathway. After systematic administration of pathway modulating drugs or drug candidates, analyses of KinCon structure dynamics may reveal alterations in potency, efficacy, and potential synergistic effects of the tested bioactive small molecules (schematic dose response curves are depicted). D) Simplified schematic representation of the activation mechanisms of BRAF, LKB1, RIPK1 and CDK6 complexes (with indication of selected regulators or complex components) engaged in altering OFF (top) or ON (bottom) kinase states. E) Representative KinCon experiments of time-dependent expressions of indicated KinCon reporter constructs in HEK293T cells are shown (mean ±SEM). Indicated KinCon reporters were transiently over-expressed in 24-well format in HEK293T cells for 10h, 16h, 24h and 48h each. Immunoblotting show expression levels of endogenous kinases and over-expressed KinCon reporters. F) Impact of 1µM PLX8394 exposure (for 1h) on BRAF and BRAF-V600E KinCon reporters (HEK293T cells) is shown. Representative of n=4 independent experiments is presented. G) RLuc PCA values have been normalized on the untreated conditions. The mean ±SEM of PLX8394 exposure on BRAF conformation opening and closing of n=4 experiments is shown. RLU, relative light units. Statistical significance for G: One-sample t-test (*p<0.05, **p<0.01, ***p<0.001)
LKB1 emanating complexes and mutation-related kinase activity conformations in intact cells.
A) Simplified view of the LKB1-complex composition which promotes AMPKα signaling via phosphorylation at position Thr172.B) Crystal structure of the LKB1-STRADα-MO25 complex (PDB code 2WTK (Zeqiraj et al. (2009a))) representing a snapshot of trimeric complex assembly. The missense mutations we have analyzed are indicated in blue (STRADα) and pale yellow and rose (LKB1). The ATP analogue AMP-PNP is depicted in light green sticks. C) Domain organization of human LKB1, STRADα and MO25 (Accession numbers: Q15831, Q7RTN6, Q9Y376) with indication of the kinase and pseudo-kinase domains (KD). Shown in red are tested missense mutations. These are summarized in the table together with their origin and assumed functions (Zubiete-Franco et al. (2019), Qing et al. (2022), Yang et al. (2019), Ui et al. (2014),Al Bakir et al. (2023), Islam et al. (2019), Boudeau et al. (2004)). D) Effect of co-expressions of indicated kinase complex components on AMPK phosphorylation (HeLa cells, 48h post transfection) (mean ±SEM, n=4 ind. experiments; 3x-Flag is indicated as flag). E) Illustration of the KinCon reporter setup for STRADα KinCon measurements: Effect of LKB1-STRADα-MO25 complex formation on the STRADα KinCon reporter opening and closing (HEK293T cells, 48h post transfection). Expression corrected signals (STRADα-KinCon) are shown (mean ±SEM, n=4 ind. experiments). F) KinCon reporter setup for LKB1 KinCon measurements: Effect of LKB1-STRADα-MO25 complex formation on the LKB1 KinCon reporter conformation. Expression corrected signals are shown (LKB1-KinCon; (HEK293T cells, 48h post transfection) (mean ±SEM, n=5 ind. experiments). G) LKB1-KinCon measurements upon co-expression of indicated proteins displaying the binding deficient STRADα mutations H231A/F233A (HF; see binding interface in Figure 2B/H). Expression corrected signals are displayed (HEK293T cells, 48h post transfection) (mean ±SEM, n=4 ind. experiments). H) Structure depiction highlights the localization of mutations conferring altered LKB1 functions. LKB1 residues K78, D176, and D194 (pale yellow sticks) are located within the catalytic cleft and in close proximity to AMP-PNP (light green sticks). I) Impact of LKB1 missense mutations (three patient mutations D176N, D194N and W308C and three ‘non-patient’ mutations K48R, R74A, K78I) on KinCon conformation changes upon co-expression of interactors. Expression corrected signals are displayed (HEK293T cells, 48h post transfection) (mean ±SEM, n=4 ind. experiments). Statistical significance for D, E, F, G, and I: One-sample t-test (*p<0.05, **p<0.01, ***p<0.001)
RIPK1 conformation dynamics.
A) Simplified schematic representations of the activation pathways for apoptosis and necroptosis. Highlighted in black is the combination treatment termed TBZ (10pg/ml TNFα, 10nM BV-6, and 20nM zVAD-FMK) that induce necroptosis. B) Domain organization of human RIPK1 (accession number: Q13546), RIPK2 (accession number: O43353), RIPK3 (accession number: Q9Y572) and MLKL (accession number: Q8NB16). C) Basal signals of indicated KinCon reporters following transient over-expression in HEK293T cells. Bars represent the RLU fold change relative to RIPK1, (expression corrected) (mean ±SD, n=5 ind. experiments). D) Time-dependent treatments using TBZ of HEK293T cells transiently expressing wt RIPK1 (left) and wt RIPK3 (right) KinCon reporters (expression corrected)(mean ±SD, n=3 ind. experiments). E) Domain organization of RIPK1 displaying missense mutation sites. F) 3D structure of RIPK1 dimers with functional mutations highlighted (PDB code: 6HHO (Wang et al. (2018))). GSK547 is depicted as brown sticks G) KinCon reporter signals with/without mutations (S14/15/166A, S14/15/166E, K45A) were measured in a HEK293T RIPK1 knock-out cell line (expression corrected)(mean ±SEM, n=5 ind. experiments). H) KinCon reporter signals of RIPK1 (patient loci: D324A, D324E, D324H, C601Y) were measured in HEK293T RIPK1 KO cells (expression corrected) (mean ±SD, n=5 ind. experiments). I) 3D structure of RIPK1 with the inhibitor GSK547, which binds to an allosteric site in close proximity to the ATP binding site (PDB code: 6HHO (Wang et al. (2018))). J) RIPK1 reporter signals with indicated mutations (described in G) upon exposure to GSK547 and Necrostatin 1µM, and the MEKi Cobimetinib (1µM, control experiment) or DMSO for 1h (mean ±SD, n=6 ind. experiments, HEK293T RIPK1 KO). Statistical significance for C to J: One-sample t-test (*p<0.05, **p<0.01, ***p<0.001)
CDK4/6 interactions and conformations.
A) Illustration of regulatory CDK4/6 interactions and Rb activation. B) Domain organization of CDK4, CDK6 and p16INK4a; tested point mutations are listed below. C) 3D structure of CDK6 in complex with p16INK4a. Crucial amino acids involved in the interaction of the two proteins are highlighted. The R31C mutant is depicted in orange. (PDB code 1BI7 (Russo et al. (1998))). D) PPI analyses of the kinases CDK4 and CDK6 with p16INK4a. Scheme illustrates CDK4/6 hetero-dimer formation with p16INK4a analyzed using a PCA RLuc PPI reporter system. PPI induces the complementation of RLuc PCA fragments promoting an increase in bioluminescence (HEK293T cells 48h of transient reporter expression). Bars represent the RLU fold change of PPI in relation to wt CDK4/6:p16INK4a (mean ±SEM, n=7 ind. experiments). E) Basal signal of CDK4/6 KinCon reporters with indicated mutations are shown (expressed for 48h in HEK293T cells; expression corrected signals) (mean ±SEM, n=6 ind. experiments). F) Quantification of alterations of CDK4/6 KinCon reporter bioluminescence signals (HEK293T, expression for 48h) upon exposure to indicated CDK4/6i (1µM) or DMSO for 3h (mean ±SEM, n=4 ind. experiments). Statistical significance for D-F: One-sample t-test (*p<0.05, **p<0.01, ***p<0.001)
Impact of small molecules and protein interactions on kinase activity conformations.
A+B) Depiction of molecular interactions of a type I 1/2 and type III kinase inhibitors with a kinase domains (N and C lobe). Impact of PLX8394, Cobimetinib GSK547 on wt and mutated versions of BRAF, RIPK1 and MEK1 KinCon reporters. 48h post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 1h (1µM) followed by Rluc PCA analyses (mean ±SEM, n=4 ind. experiments). C) Depiction of molecular interactions of a type I kinase inhibitor with a kinase domain (N and C lobe). Impact of p16-deficient binding (R31C mutation) and abemaciclib on indicated CDK6 kinase conformations. 48h post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 3h (1µM) followed by Rluc PCA analyses (mean ±SEM, n=4 ind. experiments). D) Bioluminescence measurement of PKAc wt and L206R KinCon reporters. HEK293T cells expressing the reporter were treated with 20 µM of Forskolin for 15 min followed by Rluc PCA analyses (mean ±SEM, n=4 ind. experiments). E) Kinase tree displays kinases for which KinCon reporter have been generated (red dots). The blue squares highlight the kinases for which approved drugs are available. Generated with kinhub.org/kinmap/. Statistical significance for A-D: One-sample t-test (*p<0.05, **p<0.01, ***p<0.001)
