FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes

Shubai Liu, Jin Zhang, Yang Kevin Xiang

Research output: Contribution to journalArticle

34 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)581-586
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume404
Issue number2
DOIs
StatePublished - Jan 14 2011
Externally publishedYes

Keywords

  • Adrenergic receptor
  • Cardiomyocyte
  • Fluorescence resonance energy transfer
  • Protein kinase A
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

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