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

Abeer Rababa'H, John W. Craft, Cori S. Wijaya, Fatin Atrooz, Qiying Fan, Sonal Singh, Ashley N. Guillory, Panagiotis Katsonis, Olivier Lichtarge, Bradley K. McConnell

Research output: Contribution to journalArticle

11 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)3277-3288
Number of pages12
JournalJournal of Molecular Biology
Volume425
Issue number18
DOIs
StatePublished - Sep 23 2013

    Fingerprint

Keywords

  • Hypertrophy
  • Immunoprecipitation
  • PDE4D3
  • Surface plasmon resonance
  • β-adrenergic receptor

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Rababa'H, A., Craft, J. W., Wijaya, C. S., Atrooz, F., Fan, Q., Singh, S., Guillory, A. N., Katsonis, P., Lichtarge, O., & McConnell, B. K. (2013). Protein kinase A and phosphodiesterase-4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP). Journal of Molecular Biology, 425(18), 3277-3288. https://doi.org/10.1016/j.jmb.2013.06.014