Molecular interactions of deregulated kinase circuits

Cellular membrane receptors sense environmental changes and relay the reshaped signal through spatially and temporally organized protein-protein interaction (PPI) networks. Many of these PPI are transient and occur in a certain cellular context, at particular times in development or in a diseased state. Investigations of alterations of molecular interactions are needed to reveal underlying regulatory mechanism. The decisive challenges are (i) to elucidate dynamic connections within pathological signaling systems, (ii) to directly analyze the diseased cell or tissue and (iii) to further extend the protein-interaction-network concept by integration of post translational modifications, diverse small-molecules and regulatory RNA interactions. In the proof of concept study we determined a dynamic PPI network defined by affinity- purification of endogenous and GPCR-controlled PKA complexes. Upon catecholamine- triggered and GPCR-mediated stress responses we report alterations of PPI and phosphorylation patterns of PKA complex members. We selected two unanticipated connections related to aberrant signaling: On the one hand we plan to characterize functional interactions of PKA with RNA-binding prion candidates which emerge in the pathology of neurodegenerative disorders. On the other hand we set out to investigate the involvement of PKA as a physical nexus between GPCR signaling, relevant for cancerogenesis. The network study is the conceptual basis for the characterisation of cyclic-nucleotide-dependent kinase complexes, isolated directly from human cancer tissue and from a highly proliferative metazoan organism. Hereby we aim to uncover unbiased information about the internal state of proliferative cells, tissues, and model organisms by pinpointing kinase related signaling hubs relevant for proliferation. Our ultimate goal is to generate integrated signaling maps to reveal PPI which contribute to abnormal signaling. The in-depth characterization of new kinase circuits should contribute to the identification of accessible targets for cancer therapies.

Cellular signal transmission requires the dynamic formation of spatiotemporally controlled molecular interactions. At the plasma membrane an array of more than 800 G protein- coupled receptors (GPCRs) receive, convert, amplify, and transmit incoming signals. GPCRs relay the information through intracellular signaling platforms which organize the actions of functionally interacting signaling enzymes and substrates. The list of hormone or neurotransmitter pathways that utilize the ubiquitous cAMP-sensing protein kinase A (PKA) system is expansive. This requires that the specificity, duration, and intensity of kinase responses are spatially and temporally restricted. Hereby, scaffolding proteins take the center stage for ensuring proper signal transmission. They unite second messenger sensors, activators, effectors, and kinase substrates within cellular micro-domains to precisely control and route signal propagation. Activated GPCRs team-up with intracellular scaffolding proteins to compartmentalize signal transmission. Scaffolds, such as the growing family of A kinase anchoring proteins (AKAPs), function as a physical nexus between receptors and molecular switches. Typically, these receptor-bound AKAPs recruit PKA to assemble dedicated polyvalent signaling complexes that are spatially and temporally confined. Using a proteomics approach we made the surprising discovery that an orphan GPCR which is localized in the primary cilium is a direct kinase scaffold. We confirmed the functional interaction of Gpr161 with PKA and we presented a structural explanation that it has absolute specificity for type I PKA regulatory subunits (RI). In experiments with zebrafish embryos we demonstrated that Gpr161 receptors recruit RI to primary cilia, where Hedgehog signaling is crucially involved in signal propagation. The mechanisms that control ciliogenesis have been extensively explored. We observed that cAMP elevation promotes cilia resorption. We identified a multimeric protein complex that includes two kinases and the ubiquitin proteasome system (UPS) which seems to be essential for ciliogenesis. Moreover we characterized how far PKA patient mutation and post-translational modifications contribute to PKA functions on the molecular level. We charaterized the UPS for the catalytic PKA subunits. Using PPI reporter analyses and phospho-proteomics based approaches we identified not just novel means of PKA regulations but also drug-driven consequences of RAFi exposure on the oncogenic RAS-RAF-ERK signaling pathways. Overall we present new insights how crucially regulated kinase signaling nodes are controlled through post- translational modifications and cancer-drug binding. The knowledge of the molecular mechanism of kinase regulation and perturbation on the cellular level should be relevant for generating new types of kinase inhibitors with higher efficacy and less off-target effects.