University of Wollongong
Browse

File(s) not publicly available

Implementing fluorescence anisotropy screening and crystallographic analysis to define PKA isoform-selective activation by cAMP analogs

journal contribution
posted on 2024-11-14, 22:24 authored by Simon BrownSimon Brown, Cecilia Y Cheng, S Adrian Saldanha, Jian Wu, Howard B Cottam, Banumathi Sankaran, Susan S Taylor
Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIβ, RIIα, and RIIβ. Although these isoforms all respond to cAMP, they are functionally nonredundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIβ holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIβ bound to HE33. The RIIβ structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A, and the side chains are displaced to accommodate the HE33 dipropyl groups. Comparison between cAMP-bound structures reveals that RIIβ, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent noncyclic nucleotide activators to specifically target PKA iso-holoenyzmes.

History

Citation

Brown, S. H. J., Cheng, C. Y., Saldanha, S. Adrian., Wu, J., Cottam, H. B., Sankaran, B. & Taylor, S. S. (2013). Implementing fluorescence anisotropy screening and crystallographic analysis to define PKA isoform-selective activation by cAMP analogs. ACS Chemical Biology, 8 (10), 2164-2172.

Journal title

ACS Chemical Biology

Volume

8

Issue

10

Pagination

2164-2172

Language

English

RIS ID

84447

Usage metrics

    Categories

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC