Protein kinases are dynamic molecular switches that have evolved to be only transiently activated. Kinase activity is embedded within a conserved kinase core, which is typically regulated by associated domains, linkers and interacting proteins. Moreover, protein kinases are often tethered to large macromolecular complexes to provide tighter spatiotemporal control. Thus, structural characterization of kinase domains alone is insufficient to explain protein kinase function and regulation in vivo. Recent progress in structural characterization of cyclic AMP-dependent protein kinase (PKA) exemplifies how our knowledge of kinase signalling has evolved by shifting the focus of structural studies from single kinase subunits to macromolecular complexes.

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.interaction network | molecular interactions | scaffolding function | phosphorylation | primary cilium

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Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R 2 :C 2 ). Although the RIα, RIIα, and RIIβ isoforms are well studied, little is known about RIβ. We show here that RIβ is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIβ 2 :C 2 is different from its closest homolog, RIα 2 :C 2 . The full-length RIβ 2 :C 2 crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R 1 :C 1 heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling "foci" paves the way for exploring further biological roles of PKA RIβ and establishes a paradigm for PKA signaling.structural biology | signal transduction | allostery C yclic AMP-dependent protein kinase (PKA) regulates a plethora of biological events that extend from development and differentiation to memory, ion transport, and metabolism. The inactive PKA holoenzyme is composed of a regulatory (R) subunit dimer and two catalytic (C) subunits. Binding of cAMP to the R-subunits unleashes the C-subunits, thereby allowing phosphorylation of PKA substrates. Specificity of PKA signaling is achieved in large part by the isoform diversity of its R-subunits. There are two classes of R-subunits, RI and RII, which are subclassified into α and β subtypes. Each isoform is encoded by a unique gene and is functionally nonredundant. RIα-null mice display early embryonic lethality (1), whereas RIIβ-null mice have a lean phenotype and are resistant to diet-induced diabetes (2). Specific isoforms also are expressed differently in cells and tissues. The RIβ isoform is expressed abundantly in brain and spinal cord (3-5). Hippocampal slices from RIβ-null mice show a severe deficit in long-term potentiation and also in long-term depression and memory. Although RIα protein levels are increased in these mice, the hippocampal function is not rescued, suggesting a unique role for RIβ, which is the least studied Rsubunit (6, 7). Most localization studies have focused on the differences between RI and RII and showed that RI isoforms are diffused mainly in the cytoplasm, whereas RIIs typically are associated with membranous organelles. More than 50 A kinase-anchoring proteins (AKAPs) have been identified as targeting PKA to a specific subcellular location (8). The only potential AKAP that binds preferentially to the RIβ isoform identified so far is the neurofibromatosis 2 tumor suppressor protein, merlin (9).All R isoforms share the same domain organization, which includes a docking and dimerization (D/D) domain followed by an inhibitor sequence and two cAMP-binding domains (Fig. 1E)....