Two major forms of mammalian acetyl-CoA carboxylase (EC 6.4.1.2), ACC-α and ACC-β, have been described and the sequences of the isoforms deduced. ACC-β is the predominant isoform expressed in heart and skeletal muscles, in which a major role of malonyl-CoA is probably to regulate fatty acid β-oxidation. The regulatory properties of ACC-β are incompletely defined but it is known that some cellular stresses lead to inhibition in parallel with the activation of AMP-activated protein kinase (AMP-PK). Here we examine the phosphorylation state of ACC-β within intact rat cardiac ventricular myocytes. Treatment of myocytes with the β-adrenergic agonist isoprenaline (isoproterenol) led to increased ACC-β phosphorylation that was maximal within 2 min and with 50 nM agonist. Effects of isoprenaline were revealed by the incorporation of 32P into ACC in cells incubated with [32P]Pi and also by a marked decrease (approx. 80%) in subsequent phosphorylation in vitro with cAMP-dependent protein kinase (PKA). Analysis of tryptic phosphopeptides revealed that ACC-β was phosphorylated at multiple sites by incubationin vitro with PKA or AMP-PK. Treatment of myocytes with isoprenaline affected all the major phosphorylation sites of ACC-β that were recognized in vitro by purified PKA, so that subsequent phosphorylation in vitro was greatly diminished after cell stimulation. β-Adrenergic stimulation led to decreases in cellular malonyl-CoA concentrations but no changes in kinetic properties of ACC were detected after cell homogenization and partial purification of proteins. The results suggest that: (1) ACC-β is rapidly phosphorylated at multiple sites within intact cardiac ventricular myocytes after β-adrenergic stimulation, (2) ACC-β is phosphorylated in vitro by PKA and AMP-PK at multiple sites, including at least one site accessible to each kinase, as well as kinase-selective sites, and (3) PKA is a physiologically significant ACC-β kinase.
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Research Article|
July 08 1999
Multiple-site phosphorylation of the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes: evidence that cAMP-dependent protein kinase mediates effects of β-adrenergic stimulation
Adrienne N. BOONE;
Adrienne N. BOONE
*Department of Biochemistry and Molecular Biology, Faculty of Medicine, Room 4027, Medical Block ‘A', 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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Brian RODRIGUES;
Brian RODRIGUES
†Faculty of Pharmaceutical Sciences, Room 371B, Cunningham Building, University of British Columbia, 2146 East Mall, Vancouver, British Columbia, Canada V6T 1Z3
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Roger W. BROWNSEY
Roger W. BROWNSEY
1
*Department of Biochemistry and Molecular Biology, Faculty of Medicine, Room 4027, Medical Block ‘A', 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
1To whom correspondence should be addressed (rogerb@unixg.ubc.ca).
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Publisher: Portland Press Ltd
Received:
August 10 1998
Revision Received:
April 20 1999
Accepted:
May 12 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 341 (2): 347–354.
Article history
Received:
August 10 1998
Revision Received:
April 20 1999
Accepted:
May 12 1999
Citation
Adrienne N. BOONE, Brian RODRIGUES, Roger W. BROWNSEY; Multiple-site phosphorylation of the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes: evidence that cAMP-dependent protein kinase mediates effects of β-adrenergic stimulation. Biochem J 15 July 1999; 341 (2): 347–354. doi: https://doi.org/10.1042/bj3410347
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