RESUMEN
Protein Kinase A (PKA) neuronal function is controlled by the interaction of a regulatory (R) subunit dimer to two catalytic (C) subunits. Recently, the L50R variant in the gene encoding the RIß subunit was identified in individuals with a novel neurodegenerative disease. However, the mechanisms driving the disease phenotype remained unknown. In this study, we generated a mouse model carrying the RIß-L50R mutation to replicate the human disease phenotype and study its progression with age. We examined postmortem brains of affected individuals as well as live cell cultures. Employing biochemical assays, immunohistochemistry, and behavioral assessments, we investigated the impact of the mutation on PKA complex assembly, protein aggregation and neuronal degeneration. We reveal that RIß is an aggregation-prone protein that progressively accumulates in wildtype and Alzheimer's mouse models with age, while aggregation is accelerated in the RIß-L50R mouse model. We define RIß-L50R as a causal mutation driving an age-dependent behavioral and disease phenotype in human and mouse models. Mechanistically, this mutation disrupts RIß dimerization, leading to aggregation of its monomers. Intriguingly, interaction with the C-subunit protects the RIß-L50R from self-aggregating, in a dose-dependent manner. Furthermore, cAMP signaling induces RIß-L50R aggregation. The pathophysiological mechanism elucidated here for a newly recognized neurodegenerative disease, in which protein aggregation is the result of disrupted homodimerization, sheds light on a remarkably under-appreciated but potentially common mechanism across several neurodegenerative diseases.
RESUMEN
Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.
Asunto(s)
Arginina , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Arginina/metabolismo , Humanos , Animales , Ratones , Línea Celular , Células HEK293 , Activación Enzimática/genética , Mutación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estructura Terciaria de Proteína , Modelos Moleculares , Cristalización , Secuencia de AminoácidosRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sustancias Macromoleculares/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Sustancias Macromoleculares/química , Fosforilación , Isoformas de Proteínas , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Luciferasas de Renilla , Ratones , Fosforilación , Pez CebraRESUMEN
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 RIIß isoforms are well studied, little is known about RIß. We show here that RIß 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 RIß(2):C(2) is different from its closest homolog, RIα(2):C(2). The full-length RIß(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 RIß and establishes a paradigm for PKA signaling.
Asunto(s)
Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/química , Animales , Cristalografía por Rayos X , Holoenzimas , Ratones , Estructura Cuaternaria de Proteína , Sistemas de Mensajero Secundario/fisiología , Relación Estructura-ActividadRESUMEN
Protein kinase A (PKA) signaling is essential for numerous processes but the subcellular localization of specific PKA regulatory (R) and catalytic (C) subunits has yet to be explored comprehensively. Additionally, the localization of the Cß subunit has never been spatially mapped in any tissue even though â¼50% of PKA signaling in neuronal tissues is thought to be mediated by Cß. Here we used human retina with its highly specialized neurons as a window into PKA signaling in the brain and characterized localization of PKA Cα, Cß, RIIα, and RIIß subunits. We found that each subunit presented a distinct localization pattern. Cα and Cß were localized in all cell layers (photoreceptors, interneurons, retinal ganglion cells), while RIIα and RIIß were selectively enriched in photoreceptor cells where both showed distinct patterns of co-localization with Cα but not Cß. Only Cα was observed in photoreceptor outer segments and at the base of the connecting cilium. Cß in turn, was highly enriched in mitochondria and was especially prominent in the ellipsoid of cone cells. Further investigation of Cß using RNA BaseScope technology showed that two Cß splice variants (Cß4 and Cß4ab) likely code for the mitochondrial Cß proteins. Overall, our data indicates that PKA Cα, Cß, RIIα, and RIIß subunits are differentially localized and are likely functionally non-redundant in the human retina. Furthermore, Cß is potentially important for mitochondrial-associated neurodegenerative diseases previously linked to PKA dysfunction.
RESUMEN
Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIß or the RIIß subunit results in distinct neuronal phenotypes. Although RIß 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. RIß is enriched in dendrites and co-localizes with MAP2, whereas RIIß is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIß in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIß, but not of RIIß, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.
Asunto(s)
Química Encefálica , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Isoformas de Proteínas/análisis , Animales , Axones/química , Dendritas/química , Inmunohistoquímica , RatonesRESUMEN
A-kinase anchoring proteins (AKAPs) regulate cAMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP2 (D-AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D-AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α-helix to PKA and a ß-strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D-AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D-AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C-terminus of D-AKAP2, which contains two binding motifs-the D-AKAP2AKB and the PDZ motif-that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D-AKAP2AKB binds to the D/D domain of the R-subunit and the C-terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D-AKAP2 would exhibit a differential mode of binding to the two PKA isoforms.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/química , Proteínas Portadoras/química , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Proteínas de Anclaje a la Quinasa A/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Humanos , Proteínas de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Dominios PDZ , Conformación Proteica , RatasRESUMEN
The role of the serine/threonine protein kinase, glycogen synthase kinase-3 (GSK-3), in attenuating the insulin signalling pathway has led to the concept that inhibition of GSK-3 may have therapeutic benefits in the treatment of insulin resistance and Type 2 diabetes. Indeed, various selective GSK-3 inhibitors have been developed recently and have proven to promote insulin-like effects and to act as insulin sensitisers in both in vitro and in vivo systems. GSK-3 inhibition may thus present a new, effective approach for the treatment of insulin resistance and Type 2 diabetes. This review describes the qualifications of GSK-3 as a novel drug-discovery target for Type 2 diabetes and discusses the strategies and challenges in developing small-molecule inhibitors for this important protein kinase.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Resistencia a la Insulina , Diseño de Fármacos , HumanosRESUMEN
It has been suggested that phosphorylation at serine 9 near the N-terminus of glycogen synthase kinase-3beta (GSK-3beta) mimics the prephosphorylation of its substrate and, therefore, the N-terminus functions as a pseudosubstrate. The molecular basis for the pseudosubstrate's binding to the catalytic core and autoinhibition has not been fully defined. Here, we combined biochemical and computational analyses to identify the potential residues within the N-terminus and the catalytic core engaged in autoinhibition of GSK-3beta. Bioinformatic analysis found Arg4, Arg6, and Ser9 in the pseudosubstrate sequence to be extremely conserved through evolution. Mutations at Arg4 and Arg6 to alanine enhanced GSK-3beta kinase activity and impaired its ability to autophosphorylate at Ser9. In addition, and unlike wild-type GSK-3beta, these mutants were unable to undergo autoinhibition by phosphorylated Ser9. We further show that Gln89 and Asn95, located within the catalytic core, interact with the pseudosubstrate. Mutation at these sites prevented inhibition by phosphorylated Ser9. Furthermore, the respective mutants were not inhibited by a phosphorylated pseudosubstrate peptide inhibitor. Finally, computational docking of the pseudosubstrate into the catalytic active site of the kinase suggested specific interactions between Arg6 and Asn95 and of Arg4 to Asp181 (apart from the interaction of phosphorylated serine 9 with the "phosphate binding pocket"). Altogether, our study supports a model of GSK-3-pseudosubstrate autoregulation that involves phosphorylated Ser9, Arg4, and Arg6 within the N-terminus and identified the specific contact sites within the catalytic core.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/metabolismo , Línea Celular , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Fosforilación , Especificidad por SustratoRESUMEN
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-3beta and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe67, Gln89, and Asn95 of GSK-3beta as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3beta phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3beta mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and beta-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wild-type GSK-3beta. Our studies provide important additional understanding of how GSK-3beta recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3beta residues: Phe67, Gln89, and Asn95, which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity.
Asunto(s)
Glucógeno Sintasa Quinasa 3/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas Sustrato del Receptor de Insulina , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosforilación , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , beta Catenina/metabolismoRESUMEN
Zinc is an important trace element found in most body tissues as bivalent cations and has essential roles in human health. The insulin-like effect of zinc cations raises the possibility that they inhibit glycogen synthase kinase-3beta (GSK-3beta), a serine/threonine protein kinase linked with insulin resistance and type 2 diabetes. Here we show that physiological concentrations of zinc ions directly inhibit GSK-3beta in vitro in an uncompetitive manner. Treatment of HEK-293 cells with zinc enhanced glycogen synthase activity and increased the intracellular levels of beta-catenin, providing evidence for inhibition of endogenous GSK-3beta by zinc. Moreover, zinc ions enhanced glucose uptake 3-fold in isolated mouse adipocytes, an increase similar to activation with saturated concentrations of insulin. We propose that the in vivo insulin-mimetic actions of zinc are mediated via direct inhibition of endogenous GSK-3beta.