Protein Kinase A and Phosphodiesterase-4D3 Binding to Coding Polymorphisms of Cardiac Muscle Anchoring Protein (mAKAP)

• Published in: Journal of molecular biology Vol. 425; no. 18; pp. 3277 - 3288
• Main Authors: Rababa'h, Abeer, Craft, John W., Wijaya, Cori S., Atrooz, Fatin, Fan, Qiying, Singh, Sonal, Guillory, Ashley N., Katsonis, Panagiotis, Lichtarge, Olivier, McConnell, Bradley K.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 23.09.2013
• Subjects: A Kinase Anchor Proteins - chemistry; A Kinase Anchor Proteins - genetics; A Kinase Anchor Proteins - metabolism; Amino Acid Substitution - physiology; Animals; binding sites; Binding Sites - genetics; calcium; cAMP-dependent protein kinase; CHO Cells; correlation; Cricetinae; Cricetulus; Cyclic AMP-Dependent Protein Kinases - metabolism; Cyclic Nucleotide Phosphodiesterases, Type 4 - metabolism; HEK293 Cells; Humans; hypertrophy; immunoprecipitation; mutation; myocardium; Myocardium - enzymology; PDE4D3; phosphorylation; Phosphorylation - genetics; Polymorphism, Single Nucleotide - physiology; precipitin tests; protein binding; Protein Binding - genetics; Protein Interaction Domains and Motifs - genetics; Protein Interaction Domains and Motifs - physiology; proteins; sarcoplasmic reticulum; surface plasmon resonance; Western blotting; β-adrenergic receptor; β-AR; RU; hypertrophy; AKAP; CHO; PDE4D3; PDE; CREB; GRK2; SPR; surface plasmon resonance; immunoprecipitation; mAKAP; NCBI; WT; RyR2; IBMX; PKA; PP2A; HEK; ET; NFATc; β-adrenergic receptor; PP2B; UCSC; HF; National Center for Biotechnology Information; evolutionary trace; protein phosphatase 2B, calcineurin; cAMP-response element binding; 3-isobutyl-1-methylxanthine; University of California Santa Cruz; human embryonic kidney; protein phosphatase 2A; protein kinase A; heart failure; muscle-selective A-kinase anchoring protein; nuclear factor of activated T cells; beta-adrenergic receptor; Chinese hamster ovary; G-protein coupled receptor kinase 2; A-kinase anchoring protein; response unit; wild type; phosphodiesterase; ryanodine receptors; phosphodiesterase-4D3
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2013.06.014

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 β-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, Ca2+ measurements, and Western blot. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling. [Display omitted] •mAKAP is a scaffolding protein that coordinates multi-protein signaling.•We examine the effect of naturally occurring mAKAP mutations on PKA/PDE signaling.•Human mAKAP-P1400S has lower binding propensity to PDE4D3.•Human mAKAP-S2195F and L717V mutations have enhanced PKA binding.•The mechanism may involve altered PKA substrate phosphorylation.