AKAP79 enables calcineurin to directly suppress protein kinase A activity
- University College London, United Kingdom
- KTH Royal Institute of Technology, Sweden
Abstract
Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes towards LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes.
Data availability
Source data files have been provided for figures 1-6, figure 1-supplement 2, figure 1-supplement 3, figure 3-supplement 1, and figure 3-supplement 2.Original images an uncrossed images for Coomassie-stained gels and immunoblots presented in the manuscript are shown in the zipped folder provided as an additional file.A code repository for this study may be accessed at https://github.com/jdgas/AKAP79_PKA. It contains the R code for the ABC method as well as MATLAB code for reproducing figures. The R code has to be run on a computer cluster. The repository also contains the models with a few example parameter sets, the full parameter sample as described above, and supplementary figures with simulations and experimental data for all 0, 0.2, 1 and 2 M cAMP levels with either WT S98A, or S98E RII𝛼 in the reaction mix.
Article and author information
Author details
Jeanette Hellgren Kotaleski
Funding
Wellcome Trust and Royal Society (104194/Z/14/A)
- Matthew G Gold
BBSRC (BB/N015274/1)
- Matthew G Gold
Swedish Research Council (VR-M-2017-02806)
- Matthew G Gold
European Union/Horizon 2020 (945539 Human Brain Project SGA3)
- Matthew G Gold
Erasmus Scholarship
- João Antunes
Wellcome Trust (217199/Z/19/Z)
- Saad Hannan
- Trevor G Smart
Swedish Research Council (VR-M-2020-01652)
- Matthew G Gold
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Experiments involving rats were done in accordance with the United Kingdom Animals Act, 1986 and within University College London Animal Research guidelines overseen by the UCL Animal Welfare and Ethical Review Body under project code 14058.
Copyright
© 2021, Church et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,150
- views
-
- 270
- downloads
-
- 11
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
AKAP79 enables calcineurin to directly suppress protein kinase A activity
eLife 10:e68164.
https://doi.org/10.7554/eLife.68164
Further reading
-
- Biochemistry and Chemical Biology
Identifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context is vital for capturing specific drug–protein interactions, such as those with transient protein complexes and membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target for Small molecule Target Identification Technology), a non-diffusive proximity tagging system for live cells, orthogonal to the eukaryotic system. POST-IT utilizes an engineered fusion of proteasomal accessory factor A and HaloTag to transfer Pup to proximal proteins upon directly binding to the small molecule. After significant optimization to eliminate self-pupylation and polypupylation, minimize depupylation, and optimize chemical linkers, POST-IT successfully identified known targets and discovered a new binder, SEPHS2, for dasatinib, and VPS37C as a new target for hydroxychloroquine, enhancing our understanding these drugs’ mechanisms of action. Furthermore, we demonstrated the application of POST-IT in live zebrafish embryos, highlighting its potential for broad biological research and drug development.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Inhibition of Bruton’s tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders, and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib, and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph et al., 2020). Here, we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.
