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

32 Citations (Scopus)

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

Fingerprint

Sarcoplasmic Reticulum
Cyclic AMP-Dependent Protein Kinases
Cardiac Myocytes
Chemical activation
Adenylyl Cyclases
Biosensors
Biosensing Techniques
Adrenergic Agents
Phosphorylation
Second Messenger Systems
Adrenergic Receptors
Protein Kinase Inhibitors
Occupations
Muscle Cells
Phosphotransferases
Heart Diseases
Heart Failure
Calcium

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes. / Liu, Shubai; Zhang, Jin; Xiang, Yang Kevin.

In: Biochemical and Biophysical Research Communications, Vol. 404, No. 2, 14.01.2011, p. 581-586.

Research output: Contribution to journalArticle

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