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PMID:18550536

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Citation

Manni, S, Mauban, JH, Ward, CW and Bond, M (2008) Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells. J. Biol. Chem. 283:24145-54

Abstract

Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA(cat)). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.

Links

PubMed PMC2527120 Online version:10.1074/jbc.M802278200

Keywords

Adaptor Proteins, Signal Transducing; Amino Acid Substitution; Animals; Calcium/metabolism; Calcium Signaling/physiology; Calcium-Binding Proteins/genetics; Calcium-Binding Proteins/metabolism; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cells, Cultured; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism; Enzyme Activation/genetics; Models, Biological; Mutation, Missense; Myocytes, Cardiac/enzymology; Myocytes, Cardiac/metabolism; Phosphorylation; Rats; Ryanodine Receptor Calcium Release Channel/genetics; Ryanodine Receptor Calcium Release Channel/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

RAT:KAP2

located_in

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAP2

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAP3

located_in

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAP3

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAPCA

located_in

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAPCA

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAPCB

located_in

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAPCB

located_in

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:AKA7G

enables

GO:0034237: protein kinase A regulatory subunit binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

RAT:KAPCA

part_of

GO:0005737: cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RAT:KAPCA

part_of

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

See also

References

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