FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes.
Biochem Biophys Res Commun
; 404(2): 581-6, 2011 Jan 14.
Article
em En
| MEDLINE
| ID: mdl-21130738
ABSTRACT
The second messenger cAMP-dependent protein kinase A (PKA) plays an important role in the various cellular and physiological responses. On the sarcoplasmic reticulum (SR) in cardiomyocytes, PKA regulates the calcium cycling for exciting-contraction coupling, which is often dysfunctional in a variety of heart diseases including heart failure. Here, we have developed a novel FRET-based A-kinase activity biosensor (AKAR), termed SR-AKAR3, to visualize the PKA dynamics on the SR. Activation of adrenergic receptor induces a rapid and significant increase in SR-AKAR3 FRET ratio, which is dependent on agonist occupation of the receptor and inhibited by H-89, a PKA inhibitor. Interestingly, direct activation of adenylyl cyclases or application of a cAMP analog 8-Br-cAMP induced much slower and smaller increases in SR-AKAR3 FRET ratio. These data indicate that the signaling induced by adrenergic stimulation displays a preferential access to the SR in comparison to those by direct activation of adenylyl cyclases. More, SR-AKAR3 mimics endogenous protein phospholamban on the SR for PKA-mediated phosphorylation and myocyte contraction response under adrenergic stimulation. Together, this new PKA activity biosensor provides a useful tool to directly visualize the dynamic regulation of PKA activity on the SR in cardiomyocytes under various physiological and clinical conditions.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Retículo Sarcoplasmático
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Técnicas Biossensoriais
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Proteínas Quinases Dependentes de AMP Cíclico
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Miócitos Cardíacos
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Transferência Ressonante de Energia de Fluorescência
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article