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 beta-adrenergic stimulation.
Two major forms of mammalian acetyl-CoA carboxylase (EC 6.4.1.2), ACC-alpha and ACC-beta, have been described and the sequences of the isoforms deduced. ACC-beta is the predominant isoform expressed in heart and skeletal muscles, in which a major role of malonyl-CoA is probably to regulate fatty acid beta-oxidation. The regulatory properties of ACC-beta 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-beta within intact rat cardiac ventricular myocytes. Treatment of myocytes with the beta-adrenergic agonist isoprenaline (isoproterenol) led to increased ACC-beta 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-beta was phosphorylated at multiple sites by incubation in vitro with PKA or AMP-PK. Treatment of myocytes with isoprenaline affected all the major phosphorylation sites of ACC-beta that were recognized in vitro by purified PKA, so that subsequent phosphorylation in vitro was greatly diminished after cell stimulation. beta-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-beta is rapidly phosphorylated at multiple sites within intact cardiac ventricular myocytes after beta-adrenergic stimulation, (2) ACC-beta 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-beta kinase.
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.
A N
AN
Boone
B
B
Rodrigues
R W
RW
Brownsey
D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D000318:Adrenergic beta-Agonists; D011943:Receptors, Adrenergic, beta; D017868:Cyclic AMP-Dependent Protein Kinases; D000103:Acetyl-CoA Carboxylase; D007545:Isoproterenol