Human Signaling Scaffold Protein (mAKAP) Polymorphisms: Role in Heart Failure
Rababa'H, Abeer 1981-
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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 (PP2A) to the cardiomyocytes sarcoplasmic reticulum and perinuclear space. 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 both the PKA or PDE4D3 binding domain and understanding their role in altered signaling. Our immunopreciptation study of mAKAP-P1400S, a mutation in the mAKAP-PDE4D3 binding site, displayed a significant reduction in binding affinity to PDE4D3 after stimulation, with no significant change in PKA binding and activity. Conversely, mAKAP-S2195F, a mutation located in mAKAP-PP2A binding site and flanking PKA-RII binding domain, showed significant increase in both binding affinity to PKA as well as PKA activity. Although, mAKAP-L717V (a mutation flanking the mAKAP-spectrin repeat domain) exhibited an enhanced binding propensity to PKA, it showed similar pattern of PKA activity as the wild-type mAKAP. All three mutations have similar total phosphodiesterase enzyme activity. Binding results were quantified using surface plasmon resonance (Biacore-2000). We demonstrated specific binding of wild-type mAKAP to PDE4D3. Additionally, human mAKAP mutants S2195F and L717V displayed increased expression for downstream PKA substrates and hypertrophic markers such as CREB and calcineurin. These data suggest that S2195F or L717V-mAKAP may enhance cardiac hypertrophy through persistent binding of mAKAP to PKA. Furthermore, these mutants increased the phosphorylation of ERK5 compared to the wild-type mAKAP suggesting that mAKAP also orchestrates the cross-talk of mitogen activated protein kinase (MAPK) signaling pathway with cAMP/PKA signaling pathways. Generally, PKA-PDE4D3-mAKAP complexes exemplify how protein kinases and phosphodiesterase may contribute in molecular signaling to dynamically normalize localized intracellular signaling. Consequently, comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.