Figures and data
Kinase regulation and KinCon reporter technology features.
A) Impact of indicated factors/features (e.g. Protein-protein interactions (PPI), 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 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 using either intact cell populations or lysed cells upon RLuc 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. Transient expression of the genetically encoded reporter in different 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 kinase, interlinked kinases or proteinogenic regulators of the pathway. After systematic administration of pathway modulating drugs or drug candidates, analyses of KinCon dynamics reveal changes in potency, efficacy, and potential synergistic effects of the tested bioactive small molecules. D) Shown is a simplified schematic representation of the activating 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). BRAF-V600E, LKB1, RIPK1 and CDK6 KinCon reporters were transiently over-expressed in 24-well format in HEK293T cells for 10h, 16h, 24h and 48h each. Immunoblotting with respective antibodies show expression levels of endogenous and over-expressed kinases. F) Impact of 1µM PLX8394 exposure (for 1h) on BRAF and BRAF-V600E KinCon reporters transiently over-expressed for indicated time frames in HEK293T cells is shown. One representative experiment with the respective immunoblots of in total n=4 independent performed experiments is presented. G) RLuc PCA values have been normalized on the untreated conditions for every time point. The mean ±SEM of PLX8394 exposure on BRAF dynamics 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) The crystal structure of the LKB1-STRADα-MO25 complex (PDB code 2WTK (Zeqiraj et al. (2009a))) represents a snapshot of trimeric complex assembly. The missense mutations we have analyzed are indicated in blue (STRADα) and pale yellow and rose (LKB1). The uncleavable ATP analogue AMP-PNP bound to the catalytic pocket 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 pseudokinase domains (KD). Shown in red are tested missense mutations. These are summarized in the table together with their origin and assumed functions (PM stands for patient mutation) (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 LKB1 co-expression in the presence and absence of the complex partners MO25 and STRADα on the pAMPK/AMPK ratio was determined (HeLa cells, 48h post transfection) (mean ±SEM, n=4 ind. experiments ). Representative western blots showing the presence/absence of interaction partners are shown below (3x-Flag expressed constructs 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 (HEK293T cells, 48h post transfection). Bioluminescence signals were normalized on reporter expression levels and compared to the STRADα-KinCon signal (mean ±SEM, n=4 ind. experiments). Representative western blots are shown below (3x-Flag expressed constructs indicated as flag). F) Illustration of the KinCon reporter setup for LKB1 KinCon measurements: Effect of LKB1-STRADα-MO25 complex formation on the LKB1 KinCon reporter conformation. Bioluminescence signals were measured 48h post transfection, were normalized on protein expression levels and compared to the LKB1-KinCon signal (HEK293T cells, 48h post transfection) (mean ±SEM, n=5 ind. experiments). G) LKB1-KinCon measurements upon co-expression of wt and mutated STRADα proteins displaying the binding deficient STRADα mutations H231A/F233A (HF; see binding interface in Figure 2B/H). Bioluminescence signals were normalized on protein expression levels and are displayed relative to the LKB1-KinCon signal (HEK293T cells, 48h post transfection) (mean ±SEM, n=4 ind. experiments). One representative immunoblot is shown below (3x-Flag expressed constructs indicated as flag). H) A more detailed depiction of the trimeric complex from figure 2B 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). Please note that K79 forms a salt bridge with E98 (dark green sticks) on the αC-helix. STRADα residues H231 and F233 (blue sticks) are located within the LKB1 interaction interface. LKB1 residue W308 (rose sticks) is part of a hydrophobic cluster of residues and is surrounded by the lipophilic side chains of neighboring residues (dark 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 flag-tagged STRADα and MO25. Bioluminescence signals were normalized on protein expression levels and are displayed relative to the LKB1-wt KinCon reporter (HEK293T cells, 48h post transfection) (mean ±SEM, n=4 ind. experiments). Representative immunoblots are shown below (3x-Flag expressed constructs indicated as flag). 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 chematic representations of the activation pathways for apoptosis and necroptosis are depicted. Highlighted are the compounds for the TBZ (10pg/ml TNFα, 10nM BV-6, and 20nM zVAD-FMK) treatment that induce necroptosis in black. 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, which have been normalized on the KinCon protein expression levels (mean ±SEM;, n=5 ind. experiments). D) Time-dependent treatments with TBZ of HEK293T cells transiently expressing wt RIPK1 (left) and wt RIPK3 (right) KinCon reporters. Bioluminescence signals were normalized on protein expression levels and are displayed relative to the untreated reporter signals (mean ±SEM, n=3 ind. experiments). E) Domain organization of RIPK1 with indication of missense mutation sites in RIPK1 and their suggested mode of regulation. F) 3D structure of RIPK1 dimers with functional mutations highlighted (PDB code: 6HHO (Wang et al. (2018))). The kinase-bound inhibitor GSK547 is depicted as brown sticks G) KinCon reporter signals of functional RIPK1 mutations (S14/15/166A, S14/15/166E, K45A) measured in a HEK293T RIPK1 knock-out cell line. Bioluminescence signals were normalized on KinCon protein expression levels and are displayed relative to wt-RIPK1 (mean ±SEM, n=5 ind. experiments). H) KinCon reporter signals of RIPK1 mutations originating from patient loci (D324A, D324E, D324H, C601Y) were measured in HEK293T RIPK1 KO cells. Bioluminescence signals were normalized on KinCon protein expression levels and are displayed relative to wt-RIPK1 (mean ±SEM, 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. J) Dynamics of RIPK1 reporters displaying indicated mutations (described in G) upon exposure to the two RIPK1i (GSK547 and Necrostatin 1µM), and the MEKi Cobimetinib (1µM, control experiment) or DMSO for 1h. Bars represent the RLU fold-change relative to the DMSO control of the respective reporter (mean±SEM, n=6 ind. experiments, HEK293T RIPK1 KO). Statistical significance for C, D, G, H and J: One-sample t-test (*p<0.05, **p<0.01, ***p<0.001)
CDK4/6 interactions and conformations.
A) Schematic 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 were exposed to measurements after 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. Constructs were transiently expressed for 48h in HEK293T cells. Bars represent the RLU fold change relative to the wt CDK4/6 reporters (mean ±SEM, n=6 ind. experiments). One representative western blot is shown below. F) Dynamics of CDK4/6 reporters expressed in HEK293T cells for 48h upon exposure to the three indicated CDK4/6i (1µM) or DMSO for 3h. Bars represent the RLU fold change relative to the DMSO control of the respective reporter (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 bioactive or second messenger 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 and on wt and mutated versions of BRAF, RIPK1 and MEK1 KinCon reporters. 48h after transfection of HEK293T cells with respective reporter constructs, the cells were treated with the indicated inhibitors for 1h (1µM) followed by Rluc PCA analyses. Bars represent the RLU fold change relative to the DMSO control of the respective reporter (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 after transfection of HEK293T cells with respective reporter constructs, the cells were treated with the indicated inhibitors for 3h (1µM) followed by Rluc PCA analyses. Bars represent the RLU fold change in relation to untreated wt CDK6 (mean ±SEM, n=4 ind. experiments). D) Bioluminescence measurement of PKAc wt and L206R KinCon reporters. After 48h of transient expressions in HEK293T cells, the cells were treated with 20µM of Forskolin for 15 min followed by Rluc PCA analyses. Bars represent the RLU fold change in relation to untreated wt PKAc (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 kinase 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)
