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1.
Cell Rep ; 40(2): 111073, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35830806

RESUMO

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.


Assuntos
Síndrome de Cushing , Animais , Domínio Catalítico/genética , Síndrome de Cushing/genética , Síndrome de Cushing/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hidrocortisona/metabolismo , Camundongos , Proteômica
2.
Elife ; 102021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34250905

RESUMO

Pathophysiological defects in water homeostasis can lead to renal failure. Likewise, common genetic disorders associated with abnormal cytoskeletal dynamics in the kidney collecting ducts and perturbed calcium and cAMP signaling in the ciliary compartment contribute to chronic kidney failure. We show that collecting ducts in mice lacking the A-Kinase anchoring protein AKAP220 exhibit enhanced development of primary cilia. Mechanistic studies reveal that AKAP220-associated protein phosphatase 1 (PP1) mediates this phenotype by promoting changes in the stability of histone deacetylase 6 (HDAC6) with concomitant defects in actin dynamics. This proceeds through a previously unrecognized adaptor function for PP1 as all ciliogenesis and cytoskeletal phenotypes are recapitulated in mIMCD3 knock-in cells expressing a phosphatase-targeting defective AKAP220-ΔPP1 mutant. Pharmacological blocking of local HDAC6 activity alters cilia development and reduces cystogenesis in kidney-on-chip and organoid models. These findings identify the AKAP220-PPI-HDAC6 pathway as a key effector in primary cilia development.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cílios/metabolismo , Desacetilase 6 de Histona/metabolismo , Homeostase , Rim/metabolismo , Proteína Fosfatase 1/metabolismo , Actinas/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Túbulos Renais Coletores , Camundongos , Organoides/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Proc Natl Acad Sci U S A ; 113(30): E4328-37, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27402760

RESUMO

Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an "actin barrier" that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Actinas/metabolismo , Aquaporina 2/metabolismo , Rim/metabolismo , Reabsorção Renal , Proteínas de Ancoragem à Quinase A/genética , Animais , Feminino , Homeostase , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos Knockout , Técnicas de Cultura de Órgãos , Água/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
4.
Nat Immunol ; 5(1): 88-97, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14691481

RESUMO

Microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. However, ECM components can also serve as an integral part of the innate immunity. Mice lacking expression of mindin (spondin 2), a highly conserved ECM protein, have an impaired ability to clear bacterial infection, and mindin-deficient macrophages show defective responses to a broad spectrum of microbial stimuli. Moreover, mindin binds directly to bacteria and their components and functions as an opsonin for macrophage phagocytosis of bacteria. Thus, mindin is essential in the initiation of the innate immune response and represents a unique pattern-recognition molecule in the ECM for microbial pathogens.


Assuntos
Proteínas da Matriz Extracelular/imunologia , Matriz Extracelular/imunologia , Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Sequência de Aminoácidos , Animais , Citocinas/imunologia , Citocinas/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Bactérias Gram-Positivas/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Ácidos Teicoicos/imunologia
5.
Proc Natl Acad Sci U S A ; 99(10): 6937-42, 2002 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-12011451

RESUMO

If T cells require specific interactions with MHC-bound peptides during positive selection, then the specificities of T cells selected by one peptide should be distinct from those selected by another. We have examined positive selection of CD4 T cells in four strains of mice, each overexpressing a different peptide-1-A(b)(A(b)) complex. We show that a subset of CD4 T cells is selected by the overexpressed peptide and that the specificities of the CD4 T cells, as measured by reactivity to wild-type antigen-presenting cells, vary greatly depending on which peptide is overexpressed. These differences in specificity are mediated through positive selection not negative selection. Each of the four peptide-A(b) complexes appears to adopt a different conformation, and these differences correlate with the differences in reactivity. Our results suggest that individual peptide-MHC complexes positively select different subsets of self-MHC-reactive T cells and that the conformation of the peptide-MHC complex may contribute to this process.


Assuntos
Apresentação de Antígeno/imunologia , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Moléculas de Adesão Celular , Antígenos de Histocompatibilidade Classe II/imunologia , Lectinas , Sequência de Aminoácidos , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Proteínas rab5 de Ligação ao GTP/imunologia
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