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J Cell Sci ; 130(19): 3248-3260, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28808085


Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome c oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs.

Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Linhagem Celular Transformada , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética
PLoS Biol ; 13(7): e1002192, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26158466


To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIß holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIß subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIß holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIß holoenzyme since the RIIß holoenzyme is located close to ion channels. The 2.8Å structure of an RIIßp2:C2:(Ca2ADP)2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(ß,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIß subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIß signaling cycle, we show that release of pRIIß in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIß holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.

Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Catálise , AMP Cíclico/metabolismo , Escherichia coli , Células HeLa , Holoenzimas/metabolismo , Humanos , Magnésio/metabolismo , Camundongos , Células NIH 3T3 , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ratos
Proc Natl Acad Sci U S A ; 110(5): E387-96, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23319652


cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism,and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release,and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart.

Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fator de Indução de Apoptose/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Células HeLa , Coração/fisiopatologia , Humanos , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Nucleares/genética , Oxidantes/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley
PLoS One ; 6(4): e18713, 2011 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-21556136


The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.

Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , NF-kappa B/metabolismo , Fosfotransferases/metabolismo , Linhagem Celular , Meios de Cultura Livres de Soro , Humanos , Fosforilação , Ligação Proteica , Transporte Proteico
J Cell Biol ; 193(2): 347-63, 2011 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-21502359


Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling.

Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Corpos Multivesiculares , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Transporte Proteico , Transdução de Sinais
Proc Natl Acad Sci U S A ; 102(2): 349-54, 2005 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-15630084


The genetic variability and covalent modifications associated with the amino terminus of the protein kinase A (PKA) catalytic (C) subunit suggest that it may contribute to protein-protein interactions and/or localization. By using a yeast two-hybrid screen, we identified a PKA-interacting protein (AKIP1) that binds to the amino terminus (residues 1-39) of the C subunit of PKA. The interaction was localized to the A helix (residues 14-39) of the C subunit and to the carboxyl terminus of AKIP1. AKIP1 thus defines the amino-terminal A helix of PKA as a protein interaction motif. In normal breast (Hs 578 Bst) and HeLa cells, AKIP1 is present in the nucleus as speckles. A nuclear localization signal (Arg-14 and Arg-15) was identified. Upon stimulation with forskolin, HeLa cells expressing AKIP1 accumulated higher levels of the endogenous C subunit in the nucleus. Deletion of the carboxyl terminus of AKIP1 or overexpression of residues 1-39 of the C subunit abolished nuclear localization of the activated endogenous C subunit. Thus, AKIP1 describes a PKA-interacting protein that can contribute to localization by a mechanism that is distinct from A-kinase anchoring proteins that interact with the regulatory subunits.

Transporte Ativo do Núcleo Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Nucleares/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/química , Subunidades Proteicas , Transporte Proteico , Proteínas