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 deduce d. ACC-beta is the predominant isoform expressed in heart and skeletal musc les, in which a major role of malonyl-CoA is probably to regulate fatty aci d beta-oxidation. The regulatory properties of ACC-beta are incompletely de fined but it is known that some cellular stresses lead to inhibition in par allel with the activation of AMP-activated protein kinase (AMP-PK). Here we examine the phosphorylation state of ACC-beta within intact rat cardiac ve ntricular 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 w ere revealed by the incorporation of P-32 into ACC in cells incubated with [P-32]P-1 and also by a marked decrease (approx. 80%) in subsequent phospho rylation in vitro with cAMP-dependent protein kinase (PKA). Analysis of try ptic 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 phosphorylati on in vitro was greatly diminished after cell stimulation. beta-Adrenergic stimulation fed to decreases in cellular malonyl-CoA concentrations but no changes in kinetic properties of ACC were detected after cell homogenizatio n and partial purification of proteins. The results suggest that: (1) ACC-b eta is rapidly phosphorylated at multiple sites within intact cardiac ventr icular myocytes after beta-adrenergic stimulation, (2) ACC-beta is phosphor ylated 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.