Dataset Information

Protein kinase A and phosphodiesterase-4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP).

ABSTRACT: Protein kinase A (PKA) substrate phosphorylation is facilitated through its co-localization with its signaling partner by A-kinase anchoring proteins (AKAPs). mAKAP (muscle-selective AKAP) localizes PKA and its substrates such as phosphodiesterase-4D3 (PDE4D3), ryanodine receptor, and protein phosphatase 2A (PP2A) to the sarcoplasmic reticulum and perinuclear space. The genetic role of mAKAP, in modulating PKA/PDE4D3 molecular signaling during cardiac diseases, remains unclear. The purpose of this study was to examine the effects of naturally occurring mutations in human mAKAP on PKA and PDE4D3 signaling. We have recently identified potentially important human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to ?-adrenergic receptor signaling. Three mutations (P1400S, S2195F, and L717V) were cloned and transfected into a mammalian cell line for the purpose of comparing whether those substitutions disrupt mAKAP binding to PKA or PDE4D3. Immunoprecipitation study of mAKAP-P1400S, a mutation located in the mAKAP-PDE4D3 binding site, displayed a significant reduction in binding to PDE4D3, with no significant changes in PKA binding or PKA activity. Conversely, mAKAP-S2195F, a mutation located in mAKAP-PP2A binding site, showed significant increase in both binding propensity to PKA and PKA activity. Additionally, mAKAP-L717V, a mutation flanking the mAKAP-spectrin repeat domain, exhibited a significant increase in PKA binding compared to wild type, but there was no change in PKA activity. We also demonstrate specific binding of wild-type mAKAP to PDE4D3. Binding results were demonstrated using immunoprecipitation and confirmed with surface plasmon resonance (Biacore-2000); functional results were demonstrated using activity assays, Ca(2+) measurements, and Western blot. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.

SUBMITTER: Rababa'h A

PROVIDER: S-EPMC3808176 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Protein kinase A and phosphodiesterase-4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP).

Rababa'h Abeer A Craft John W JW Wijaya Cori S CS Atrooz Fatin F Fan Qiying Q Singh Sonal S Guillory Ashley N AN Katsonis Panagiotis P Lichtarge Olivier O McConnell Bradley K BK

Journal of molecular biology 20130625 18

Protein kinase A (PKA) substrate phosphorylation is facilitated through its co-localization with its signaling partner by A-kinase anchoring proteins (AKAPs). mAKAP (muscle-selective AKAP) localizes PKA and its substrates such as phosphodiesterase-4D3 (PDE4D3), ryanodine receptor, and protein phosphatase 2A (PP2A) to the sarcoplasmic reticulum and perinuclear space. The genetic role of mAKAP, in modulating PKA/PDE4D3 molecular signaling during cardiac diseases, remains unclear. The purpose of th ...[more]

PMID: 23806656

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Project description:BackgroundCardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.ResultsDuring a yeast 2-hybrid (Y2H) library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL) as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP).To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor.We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage.ConclusionsThis study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL is an important regulator of cardiac contractility. This has further implications for understanding the patho-aetiology of HCM-causing mutations in the genes encoding cMyBPC and cTNI, and raises the question of whether MMGL might itself be considered a candidate HCM-causing or modifying factor.

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.

Dataset Information

Protein kinase A and phosphodiesterase-4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP).

Publications

Protein kinase A and phosphodiesterase-4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP).

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