BACKGROUND
Stress signaling typically involves stimulation of the β-adrenergic pathway which
leads to the subsequent activation of cAMP-dependent protein kinase A (PKA). PKA has
thus been proposed to regulate calcium entry into cardiomyocytes during stress signaling
via the L-type calcium channel (CaV1.2) and the ryanodine receptor 2 (RyR2). Ryanodine
Receptors (RyRs) are huge intracellular ion channels, located in the sarcoplasmic
and endoplasmic reticulum, where they control the release of stored calcium ions whereas
CaV1.2 channel is located on the plasma membrane. Both channels are targeted by disease-causing
mutations and are under strict regulation by auxiliary proteins, small molecules,
and post-translational modifications (PTMs). PTMs of these channels is a cornerstone
of their physiological and pathophysiological regulation; and aberrant phosphorylation
has been implicated in a multitude of disorders ranging from heart failure to Alzheimer's
disease. Despite decades of research, the molecular mechanism underlying this modulation
is enigmatic with multiple sites being implicated. Further there is some controversy
regarding which sites are being phosphorylated by PKA and which are important for
β-adrenergic responses.
METHODS AND RESULTS
Using high-resolution structures determined via X-ray crystallography, we show how
the catalytic domain of PKA engages both the RyR2 and CaV1.2 channels, highlighting
the target sites that are preferentially oriented for phosphoryl transfer. Using isothermal
titration calorimetry (ITC) and kinase assays, we determined the binding affinities
and enzymatic activities of PKA to various RyR2 and CaV1.2 substrates.
CONCLUSION
RyR2 domain engages PKA by wrapping around the large-lobe of its catalytic subunit,
resulting in an extensive interface not seen in PKA complexes with isolated peptides.
We also trapped complexes in both closed and open forms PKA, showing that the RyR2
substrate can bind prior to closing of PKA. The interface is targeted by multiple
disease-associated mutations that can affect the interaction and catalytic activity.
Finally, we found when one site is phosphorylated in RyR2, another site is more likely
to be phosphorylated by PKA, which can lead to amplification of the stress signal.
We also determined the X-ray crystal structure of a C-terminal peptide from CaV1.2
containing S1981 in complex with PKA. We measured a dissociation constant of 35 uM
toward this peptide via ITC, which is comparable to other PKA substrates. Our kinase
assays corroborated this finding with strong activity towards S1981. In contrast,
the affinity and enzymatic activity of PKA towards S1718 is substantially lower despite
being implicated as an important PKA phosphorylation site.
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© 2021 Published by Elsevier Inc.
