Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1–related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

Significance: At the plasma membrane an array of more than 800 G protein-coupled receptors (GPCRs) receive, convert, amplify, and transmit incoming signals. Activated GPCRs team-up with intracellular scaffolding proteins to compartmentalize signal transmission. Scaffolds, such as β-arrestin and A-kinase anchoring proteins (AKAPs), function as a physical nexus between receptors and molecular switches. Typically, these receptor-bound AKAPs recruit protein kinase A (PKA) to assemble dedicated polyvalent signaling complexes that are spatially and temporally confined. Here, we report that the orphan GPCR, Gpr161, is a PKA substrate and also has an AKAP motif embedded in its C-terminal tail. Our results suggest that Gpr161, by directly recruiting type I PKA holoenzymes to the receptor, creates a cAMP-sensing signalosome. Furthermore, we propose that Gpr161 plays a role in recruiting isoform-specific PKA complexes to primary cilia.

Compartmentalization of diverse types of signaling molecules contributes to the precise coordination of signal propagation. The primary cilium fulfills this function by acting as a spatiotemporally confined sensory signaling platform. For the integrity of ciliary signaling, it is mandatory that the ciliary signaling pathways are constantly attuned by alterations in both oscillating small molecules and the presence or absence of their sensor/effector proteins. In this context, ciliary G protein-coupled receptor (GPCR) pathways participate in coordinating the mobilization of the diffusible second messenger molecule 3′,5′-cyclic adenosine monophosphate (cAMP). cAMP fluxes in the cilium are primarily sensed by protein kinase A (PKA) complexes, which are essential for the basal repression of Hedgehog (Hh) signaling. Here, we describe the dynamic properties of underlying signaling circuits, as well as strategies for second messenger compartmentalization. As an example, we summarize how receptor-guided cAMP-effector pathways control the off state of Hh signaling. We discuss the evidence that a macromolecular, ciliary-localized signaling complex, composed of the orphan GPCR Gpr161 and type I PKA holoenzymes, is involved in antagonizing Hh functions. Finally, we outline how ciliary cAMP-linked receptor pathways and cAMP-sensing signalosomes may become targets for more efficient combinatory therapy approaches to counteract dysregulation of Hh signaling.

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation‐oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient‐mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho‐transfer. Such non‐catalytic kinase functions are related to transitions of full‐length kinase conformations. Recent findings underline that cell‐based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live‐cell recording of kinase states. KinCon is a genetically encoded bioluminescence‐based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full‐length kinase‐targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

Significance: The pharmaceutical targeting of mutated BRAF has shown promising clinical outcomes in patients with melanoma. However, more than 300 reported BRAF patient mutations and the occurrence of a kinase-drug resistance mechanism hamper patient-oriented therapies. We have developed an extendable and cell-based kinase conformation reporter platform (KinCon) to predict and compare the effect of kinase drugs. We systematically evaluated drug efficacies by assessing KinCon reporter dynamics in response to different drugs and patient mutations. Our findings suggest that FDA-approved melanoma inhibitors may have the potential to block non-V600E–mutated BRAF activities also in non–small-cell lung cancers (NSCLC). We assume that widespread analyses of KinCon:drug interactions may assist in the future in identifying patient mutation–specific and thus more effective kinase inhibitors.

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.