Abstract
Protein kinase A anchoring proteins (AKAPs) tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The muscle AKAP, mAKAP, co-localizes with the sarcoplasmic reticulum Ca2+ release channel or ryanodine receptor (RyR). The purpose of this study was to determine whether anchoring of PKA by mAKAP regulates RyR function. Either mAKAP or mAKAP-P, which is unable to anchor PKA, was expressed in CHO cells stably expressing the skeletal muscle isoform of RyR (CHO-RyR1). Immunoelectron microscopy showed that mAKAP co-localized with RyR1 in disrupted skeletal muscle. Following the addition of 10 microm forskolin to activate adenylyl cyclase, RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP increased by 42.4 +/- 6.6% (n = 4) compared with cells expressing mAKAP-P. Forskolin treatment alone did not increase the amplitude of the cytosolic Ca2+ transient in CHO-RyR1 cells expressing mAKAP or mAKAP-P; however, forskolin plus 10 mm caffeine elicited a cytosolic Ca2+ transient, the amplitude of which increased by 22% (p < 0.05) in RyR1/mAKAP-expressing cells compared with RyR1/mAKAP-P-expressing cells. Therefore, localization of PKA by mAKAP at RyR1 increases both PKA-dependent RyR phosphorylation as well as efflux of Ca2+ through the RyR. Therefore, RyR1 function is regulated by mAKAP targeting of PKA, implying an important functional role for PKA phosphorylation of RyR in skeletal muscle.
Highlights
Cyclic AMP-dependent protein kinase (PKA)1 has a wide range of substrates and elicits a variety of cellular responses
It is predicted that upon activation of the PKA pathway, PKA, which is tethered to an AKAP in the vicinity of local increases in cAMP, will preferentially be activated, compared with PKA not anchored at these sites
We first determined at the electron microscope level whether mAKAP is co-localized with ryanodine receptor (RyR) in native skeletal muscle. mAKAP is localized to two different subcellular regions
Summary
Cyclic AMP-dependent protein kinase (PKA) has a wide range of substrates and elicits a variety of cellular responses. We previously demonstrated that upon isoproterenol stimulation, PKA-dependent phosphorylation of two myofibrillar proteins, troponin I and myosin-binding protein C, was significantly reduced in cardiac myocytes expressing Ht31 [4], a peptide that binds the regulatory subunit (RII) of type II PKA and prevents AKAP-PKA interactions [5]. PKA anchoring was required in order to maintain AMPA (␣-amino3-hydroxy-5-methyl-4-isoxazole proprionic acid) responsive glutamate receptor currents in cultured hippocampal neurons [8]. Both insulin secretion and increased intracellular Ca2ϩ are inhibited in pancreatic islet cells following treatment with Ht31, and cell-permeant forms of Ht31 inhibit PKA-dependent sperm motility [9, 10]. AKAP-targeted PKA Regulates Ryanodine Receptor Function tion of the interaction between AKAP79 and the 2-AR attenuates downstream activation of the mitogen-activated protein kinase pathway [2, 11]
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