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)....

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIb or the RIIb subunit results in distinct neuronal phenotypes. Although RIb contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIb is enriched in dendrites and co-localizes with MAP2, whereas RIIb is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIb in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIb, but not of RIIb, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

Substrate recognition and specificity are essential for the reliability and fidelity of protein kinase function. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates. However, how the enzyme selects its phosphorylated substrates is unknown. Here, we combined in silico modeling with mutagenesis and biological studies to identify GSK-3-substrate interaction sites located within its binding cleft. Protein-protein docking of GSK-3␤ and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe 67 , Gln 89 , and Asn 95 of GSK-3␤ as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3␤ phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3␤ mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and ␤-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wildtype GSK-3␤. Our studies provide important additional understanding of how GSK-3␤ recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3␤ residues: Phe 67 , Gln 89 , and Asn 95 , which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity. Glycogen synthase kinase 3 (GSK-3)4 is a ubiquitous serine/ threonine kinase expressed as two isoforms (␣ and ␤) (1), and has been implicated in many biological processes, including glucose metabolism, cell apoptosis, and embryonic development (reviewed in Refs. 2-4). The cellular activity of GSK-3 is stringently controlled in response to growth factors and hormones. However, unlike most protein kinases, GSK-3 is constitutively active in resting cells and becomes inhibited upon stimulation of the cells. This inhibition is achieved through direct phosphorylation of N-terminal serine residues (Ser 21 or Ser 9 in ␣, ␤, respectively) by several protein kinases, such as PKB, p90RSK, PKA, and PKC (2-4). GSK-3 also may be phosphorylated on Tyr 216 located in the activation loop (5). This phosphorylation is an autophosphorylation event as demonstrated by in vitro and in vivo cell systems (5-7).Elevated activity of GSK-3 is associated with several diseases, including type 2 diabetes, neurodegenerative diseases, and affective disorders (8 -10). Hence, selective inhibitors of GSK-3 may be of therapeutic value and are currently under extensive development (11)(12)(13)(14). Thus, understanding of how GSK-3 interacts with its substrates may pave the way for design and development of new specific substrate competitive GSK-3 inhibitors.Substrate specificity of protein kinases is a fundamental determinant for the integrity and fidelity of signaling pathways. Previous studies formulated consensus sequences for optimal phosphorylation motifs of protein kinases usi...

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