RESUMO
Adrenergic stimulation of adipocytes yields a cAMP signal that activates protein kinase A (PKA). PKA phosphorylates perilipin, a protein localized on the surface of lipid droplets that serves as a gatekeeper to regulate access of lipases converting stored triglycerides to free fatty acids and glycerol in a phosphorylation-dependent manner. Here, we report a new function for optic atrophy 1 (OPA1), a protein known to regulate mitochondrial dynamics, as a dual-specificity A-kinase anchoring protein associated with lipid droplets. By a variety of protein interaction assays, immunoprecipitation and immunolocalization experiments, we show that OPA1 organizes a supramolecular complex containing both PKA and perilipin. Furthermore, by a combination of siRNA-mediated knockdown, reconstitution experiments using full-length OPA1 with or without the ability to bind PKA or truncated OPA1 fused to a lipid droplet targeting domain and cellular delivery of PKA anchoring disruptor peptides, we demonstrate that OPA1 targeting of PKA to lipid droplets is necessary for hormonal control of perilipin phosphorylation and lipolysis.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , GTP Fosfo-Hidrolases/fisiologia , Metabolismo dos Lipídeos/genética , Lipólise/efeitos dos fármacos , Células 3T3-L1 , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ancoragem à Quinase A/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Isoproterenol/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipólise/genética , Camundongos , Modelos Biológicos , Perilipina-1 , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta/fisiologiaRESUMO
Protein kinase A (PKA) is targeted to distinct subcellular localizations by specific protein kinase A anchoring proteins (AKAPs). AKAPs are divided into subclasses based on their ability to bind type I or type II PKA or both. Dual-specificity AKAPs were recently reported to have an additional PKA binding determinant called the RI specifier region. A bioinformatic search with the consensus RI specifier region identified a novel AKAP, the splicing factor arginine/serine-rich 17A (SFRS17A). Here, we show by a variety of protein interaction assays that SFRS17A binds both type I and type II PKA in vitro and inside cells, demonstrating that SFRS17A is a dual-specific AKAP. Moreover, immunofluorescence experiments show that SFRS17A colocalizes with the catalytic subunit of PKA as well as the splicing factor SC35 in splicing factor compartments. Using the E1A minigene splicing assay, we found that expression of wild type SFRS17A conferred regulation of E1A alternative splicing, whereas the mutant SFRS17A, which is unable to bind PKA, did not. Our data suggest that SFRS17A is an AKAP involved in regulation of pre-mRNA splicing possibly by docking a pool of PKA in splicing factor compartments.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Processamento Alternativo , Animais , Bovinos , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática , Humanos , Proteínas Nucleares/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Processamento de Serina-Arginina , Ressonância de Plasmônio de SuperfícieRESUMO
We have reported previously the design of a RIAD (RI-anchoring disruptor) peptide that specifically displaces PKA (protein kinase A) type I from the AKAP (A-kinase-anchoring protein) ezrin, which is present in the immunological synapse of T-cells. This increases immune reactivity by reducing the threshold for activation and may prove a feasible approach for improving immune function in patients with cAMP-mediated T-cell dysfunction. However, the use of RIAD in biological systems is restricted by its susceptibility to enzymatic cleavage and, consequently, its short half-life in presence of the ubiquitous serum peptidases. In the present study, carefully selected non-natural amino acids were employed in the design of RIAD analogues with improved stability. The resulting peptidomimetics demonstrated up to 50-fold increased half-lives in serum compared with RIAD, while maintaining similar or improved specificity and potency with respect to disruption of PKA type I-AKAP interactions.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteína Tirosina Quinase CSK , Bovinos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Humanos , Interleucina-2/metabolismo , Camundongos , Peptídeos/química , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Quinases da Família srcRESUMO
A-kinase anchoring proteins (AKAPs) target protein kinase A (PKA) to a variety of subcellular locations. Conventional AKAPs contain a 14-18-amino acid sequence that forms an amphipathic helix that binds with high affinity to the regulatory (R) subunit of PKA type II. More recently, a group of dual specificity AKAPs has been classified on the basis of their ability to bind the PKA type I and the PKA type II isozymes. In this study we show that dual specificity AKAPs contain an additional PKA binding determinant called the RI Specifier Region (RISR). A variety of protein interaction assays and immunoprecipitation and immunolocalization experiments indicates that the RISR augments RI binding in vitro and inside cells. Cellular delivery of the RISR peptide uncouples RI anchoring to Ezrin leading to release of T cell inhibition by cAMP. Likewise, expression of mutant Ezrin forms where RI binding has been abrogated by substitution of the RISR sequence prevents cAMP-mediated inhibition of T cell function. Thus, we propose that the RISR acts in synergy with the amphipathic helix in dual specificity anchoring proteins to enhance anchoring of PKA type I.