scholarly journals 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

1999 ◽  
Vol 341 (2) ◽  
pp. 347-354 ◽  
Author(s):  
Adrienne N. BOONE ◽  
Brian RODRIGUES ◽  
Roger W. BROWNSEY
Keyword(s):  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Acetyl Coa Carboxylase ◽  
Adrenergic Stimulation ◽  
Dependent Protein Kinase ◽  
Acetyl Coa ◽  
Camp Dependent Protein Kinase ◽  
Malonyl Coa ◽  
Dependent Protein

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.

scholarly journals Evidence that glucagon-mediated inhibition of acetyl-CoA carboxylase in isolated adipocytes involves increased phosphorylation of the enzyme by cyclic AMP-dependent protein kinase

1985 ◽  
Vol 226 (1) ◽  
pp. 139-145 ◽  
Author(s):  
R Holland ◽  
D G Hardie ◽  
R A Clegg ◽  
V A Zammit
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinetic Parameters ◽  
Acetyl Coa Carboxylase ◽  
Response Curves ◽  
Dependent Protein Kinase ◽  
Acetyl Coa ◽  
Dependent Protein ◽  
Isolated Adipocytes

The kinetic parameters and phosphorylation state of acetyl-CoA carboxylase were analysed after purification of the enzyme by avidin-Sepharose chromatography from extracts of isolated adipocytes treated with glucagon or adrenaline. The results provide evidence that the mechanism of inhibition of acetyl-CoA carboxylase in adipocytes treated with glucagon [Zammit & Corstorphine (1982) Biochem. J. 208, 783-788] involves increased phosphorylation of the enzyme. Hormone treatment had effects on the kinetic parameters of the enzyme similar to those of phosphorylation of the enzyme in vitro by cyclic AMP-dependent protein kinase. Glucagon treatment of adipocytes led to increased phosphorylation of acetyl-CoA carboxylase in the same chymotryptic peptide as that containing the major site phosphorylated on the enzyme by purified cyclic AMP-dependent protein kinase in vitro [Munday & Hardie (1984) Eur. J. Biochem. 141, 617-627]. The dose-response curves for inhibition of enzyme activity and increased phosphorylation of the enzyme were very similar, with half-maximal effects occurring at concentrations of glucagon (0.5-1 nM) which are close to the physiological range. In general, the patterns of increased 32P-labelling of chymotryptic peptides induced by glucagon or adrenaline were similar, although there were quantitative differences between the effects of the two hormones on individual peptides. The results are discussed in terms of the possible roles of cyclic AMP-dependent and -independent protein kinases in the regulation of acetyl-CoA carboxylase activity and of lipogenesis in white adipose tissue.

scholarly journals Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells

1998 ◽  
Vol 140 (1) ◽  
pp. 131-141 ◽  
Author(s):  
Helena Melander Gradin ◽  
Niklas Larsson ◽  
Ulrica Marklund ◽  
Martin Gullberg
Keyword(s):  
Protein Kinase ◽  
Genetic System ◽  
Cell Surface Receptor ◽  
In Vitro Assays ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Camp Dependent Protein Kinase ◽  
Oncoprotein 18 ◽  
Dependent Protein

Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.

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