Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-protein interactions.

Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signaling axis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition.

Enhanced TROSY Effect in [2-19 F, 2-13 C] Adenosine and ATP Analogs Facilitates NMR Spectroscopy of Very Large Biological RNAs in Solution.

Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2-19 F, 2-13 C]-adenosine, which allow the incorporation of aromatic 19 F-13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2-19 F, 2-13 C]-adenosine-phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2-19 F, 2-13 C]-adenosine triphosphate, the 124 nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2-19 F, 2-13 C]-A-labeled RNA featured sharper resonances than the [2-1 H, 2-13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2-19 F, 2-13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2-19 F, 2-13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells.

Disruptor: Computational identification of oncogenic mutants disrupting protein-protein and protein-DNA interactions.

We report an Osprey-based computational protocol to prospectively identify oncogenic mutations that act via disruption of molecular interactions. It is applicable to analyse both protein-protein and protein-DNA interfaces and it is validated on a dataset of clinically relevant mutations. In addition, it is used to predict previously uncharacterised patient mutations in CDK6 and p16 genes, which are experimentally confirmed to impair complex formation.

Tracking and blocking interdependencies of cellular BRAF-MEK oncokinase activities.

The selective targeting of mutated kinases in cancer therapies has the potential to improve therapeutic success and thereby the survival of patients. In the case of melanoma, the constitutively active MAPK pathway is targeted by a combinatorial inhibition of BRAF and MEK activities. These MAPK pathway players may display patient-specific differences in the onco-kinase mutation spectrum, which needs to be considered for the design of more efficient personalized therapies. Here, we extend a bioluminescence-based kinase conformation biosensor (KinCon) to allow for live-cell tracking of interconnected kinase activity states. First, we show that common MEK1 patient mutations promote a structural rearrangement of the kinase to an opened and active conformation. This effect was reversible by the binding of MEK inhibitors to mutated MEK1, as shown in biosensor assays and molecular dynamics simulations. Second, we implement a novel application of the KinCon technology for tracking the simultaneous, vertical targeting of the two functionally linked kinases BRAF and MEK1. Thus, we demonstrate that, in the presence of constitutively active BRAF-V600E, specific inhibitors of both kinases are efficient in driving MEK1 into a closed, inactive conformation state. We compare current melanoma treatments and show that combinations of BRAFi and MEKi display a more pronounced structural change of the drug sensor than the respective single agents, thereby identifying synergistic effects among these drug combinations. In summary, we depict the extension of the KinCon biosensor technology to systematically validate, anticipate, and personalize tailored drug arrangements using a multiplexed setup.

Tracking mutation and drug-driven alterations of oncokinase conformations.

Numerous kinases act as central nodes of cellular signaling networks. As such, many of these enzymes function as molecular switches for coordinating spatiotemporal signal transmission. Typically, it is the compartmentalized phosphorylation of protein substrates which relays the transient input signal to determine decisive physiological cell responses. Genomic alterations affect kinase abundance and/or their activities which contribute to the malignant transformation, progression, and metastasis of human cancers. Thus, major drug discovery efforts have been made to identify lead molecules targeting clinically relevant oncokinases. The concept of personalized medicine aims to apply the therapeutic agent with the highest efficacy towards a patient-specific mutation. Here, we discuss the implementation of a cell-based reporter system which may foster the decision-making process to identify the most promising lead-molecules. We present a modular kinase conformation (KinCon) biosensor platform for live-cell analyses of kinase activity states. This biosensor facilitates the recording of kinase activity conformations of the wild-type and the respective mutated kinase upon lead molecule exposure. We reflect proof-of-principle studies demonstrating how this technology has been extended to profile drug properties of the full-length kinases BRAF and MEK1 in intact cells. Further, we pinpoint how this technology may open new avenues for systematic and patient-tailored drug discovery efforts. Overall, this precision-medicineoriented biosensor concept aims to determine kinase inhibitor specificity and anticipate their drug efficacies.

Allosteric Kinase Inhibitors Reshape MEK1 Kinase Activity Conformations in Cells and In Silico.

Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK's catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting.

Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression.

Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4-induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8+ T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. SIGNIFICANCE: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression.