Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase.

Autor: Yoo R; Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Institute, Vancouver, British Columbia, Canada., Haji-Ghassemi O; Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Institute, Vancouver, British Columbia, Canada. Electronic address: omid.hajighassemi@ucalgary.ca., Bader M; Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Institute, Vancouver, British Columbia, Canada., Xu J; Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Institute, Vancouver, British Columbia, Canada., McFarlane C; Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Institute, Vancouver, British Columbia, Canada., Van Petegem F; Department of Biochemistry and Molecular Biology, University of British Columbia, Life Sciences Institute, Vancouver, British Columbia, Canada. Electronic address: filip.vanpetegem@ubc.ca.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2024 Nov 29; Vol. 301 (1), pp. 108039. Date of Electronic Publication: 2024 Nov 29.
DOI: 10.1016/j.jbc.2024.108039
Abstrakt: β-Adrenergic signaling activates cAMP-dependent PKA, which regulates the activity of L-type voltage-gated calcium channels such as Ca V 1.2. Several PKA target sites in the C-terminal tail of Ca V 1.2 have been identified, and their phosphorylation has been suggested to increase currents in specific tissues or heterologous expression systems. However, augmentation of Ca V 1.2 currents in the heart is instead mediated by phosphorylation of Rad, a small GTPase that can inhibit Ca V 1.2. It is unclear how each of the proposed target sites in Ca V 1.2 and Rad rank toward their recognition by PKA, which could reveal a preferential phosphorylation. Here, we used quantitative assays on three Ca V 1.2 and four Rad sites. Isothermal titration calorimetry and enzyme kinetics show that there are two Tiers of targets, with Ca V 1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming tier one substrates for PKA. These share a common feature with two Arginine residues at specific positions that can anchor the peptide into the substrate binding cleft of PKA. In contrast, PKA shows minimal activity for the other, tier two substrates, characterized by low k cat values and undetectable binding via isothermal titration calorimetry. The existence of two tiers suggests that PKA regulation of the Ca V 1.2 complex may occur in a graded fashion. We report crystal structures of the PKA catalytic subunit with and without a Ca V 1.2 and test the importance of several anchoring residues via mutagenesis. Different target sites utilize different anchors, highlighting the plasticity of PKAc to recognize substrates.
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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