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A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

ABSTRACT: A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by ?2-adrenergic receptors (?(2)ARs); such compartmentalization ensures speed and fidelity of cAMP signaling and effects on cell function. This study aimed to assess the role of AKAPs in regulating global and compartmentalized ?(2)AR signaling in human airway smooth muscle (ASM). Transcriptome and proteomic analyses were used to characterize AKAP expression in ASM. Stable expression or injection of peptides AKAP-IS or Ht31 was used to disrupt AKAP-PKA interactions, and global and compartmentalized cAMP accumulation stimulated by ?-agonist was assessed by radioimmunoassay and membrane-delineated flow through cyclic nucleotide-gated channels, respectively. ASM expresses multiple AKAP family members, with gravin and ezrin among the most readily detected. AKAP-PKA disruption had minimal effects on whole-cell cAMP accumulation stimulated by ?-agonist (EC(50) and B(max)) concentrations, but significantly increased the duration of plasma membrane-delineated cAMP (?=251±51 s for scrambled peptide control vs. 399±79 s for Ht31). Direct PKA inhibition eliminated decay of membrane-delineated cAMP levels. AKAPs coordinate compartmentalized cAMP signaling in ASM cells by regulating multiple elements of ?(2)AR-mediated cAMP accumulation, thereby representing a novel target for manipulating ?(2)AR signaling and function in ASM.

SUBMITTER: Horvat SJ

PROVIDER: S-EPMC3425821 | biostudies-other | 2012 Sep

REPOSITORIES: biostudies-other

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A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

Horvat Sarah J SJ Deshpande Deepak A DA Yan Huandong H Panettieri Reynold A RA Codina Juan J DuBose Thomas D TD Xin Wenkuan W Rich Thomas C TC Penn Raymond B RB

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20120530 9

A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by β2-adrenergic receptors (β(2)ARs); such compartmentalization ensures speed and fidelity of cAMP signaling and effects on cell function. This study aimed to assess the role of AKAPs in regulating global and compartmentalized β(2)AR signaling in human airway smooth muscle (ASM). Transcriptome and proteomic analyses were used to characterize AKAP expression in AS ...[more]

PMID: 22649031

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Project description:To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GR(Ser211)), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GR(Ser211) phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GR(Ser211) phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma.

Project description:One of the crucial cardiac signaling pathways is cAMP-mediated PKA signal transduction, which is regulated by a family of scaffolding proteins, i.e., A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) partly regulates cardiac cAMP/PKA signaling by binding to PKA and phosphodiesterase 4D3 (PDE4D3), among other proteins, and plays a central role in modulating cardiac remodeling. Moreover, genetics plays an incomparable role in modifying the risk of cardiovascular diseases (CVDs). Single-nucleotide polymorphisms (SNPs) in various proteins have especially been shown to predispose individuals to CVDs. Hence, we hypothesized that human mAKAP polymorphisms found in humans with CVDs alter the cAMP/PKA pathway, influencing the susceptibility of individuals to CVDs. Our computational analyses revealed two mAKAP SNPs found in cardiac disease-related patients with the highest predicted deleterious effects, Ser 1653 Arg (S1653R) and Glu 2124 Gly (E2124G). Coimmunoprecipitation data in human embryonic kidney-293T cells showed that the S1653R SNP, present in the PDE4D3-binding domain of mAKAP, changed the binding of PDE4D3 to mAKAP and that the E2124G SNP, flanking the 3'-PKA binding domain, changed the binding of PKA before and after stimulation with isoproterenol. These SNPs significantly altered intracellular cAMP levels, global PKA activity, and cytosolic PDE activity compared with the wild type before and after isoproterenol stimulation. PKA-mediated phosphorylation of pathological markers was found to be upregulated after cell stimulation in both mutants. In conclusion, human mAKAP polymorphisms may influence the propensity of developing CVDs by affecting cAMP/PKA signaling, supporting the clinical significance of PKA-mAKAP-PDE4D3 interactions. NEW & NOTEWORTHY We found that single-nucleotide polymorphisms in muscle-specific A-kinase anchoring protein found in human patients with cardiovascular diseases significantly affect the cAMP/PKA signaling pathway. Our results showed, for the first time, that human muscle-specific A-kinase anchoring protein polymorphisms might alter the susceptibility of individuals to develop cardiovascular diseases with known underlying molecular mechanisms.

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A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

Publications

A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

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