HORMONAL REGULATION OF ATP CITRATE LYASE PHOSPHORYLATION IN 3T3-L1 ADIPOCYTES

Abstract

ATP citrate lyase was purified to homogeneity form murine liver. In vitro the purified enzyme served as a good substrate for the catalytic subunit of cAMP-dependent protein kinase, accepting up to one mol of covalently bound phosphate per mol 110,000 dalton subunit. cGMP-dependent protein kinase also catalyzed the phosphorylation of lyase. High-titer anti-ATP citrate lyase serum was produced in rabbits and was employed as a specific probe for studying the hormonal regulation of the phosphorylation of ATP citrate lyase in 3T3-L1 adipocytes. Treatment of the cells with either insulin (0.18 (mu)M) or isoproterenol (10 (mu)M) plus methylisobutylxanthine (0.2 mM) stimulated the incorporation of ('32)P(,i) into lyase approximately 3-fold. A combination of optimal concentrations of both hormones also produced a 3-fold elevation in the labeling of 3T3-L1 adipocyte ATP citrate lyase suggesting a lack of additivity in the effects of the lipogenic and lipolytic hormones. Sites phosphorylated in the presence and absence of hormones were compared by complete tryptic digestion of immunoprecipitated ('32)P-ATP citrate lyase and subsequent reverse-phase HPLC and two-dimensional mapping on thin layers of cellulose. These analyses revealed that the basal, insulin-stimulated and isoproterenol-stimulated phosphorylations in 3T3-L1 adipocyte ATP citrate lyase all occurred on a single peptide. The same peptide was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase and cGMP-dependent protein kinase. The peptide was not digested further by chymotrypsin or protease V.8, or cleaved by cyanogen bromide but was digested by pronase. Utilizing digestion by DAP-I, the site of phosphorylation in response to insulin and isoproterenol in 3T3-L1 adipocytes was localized to a single dipeptide.;The M(,r) of the phosphopeptide was estimated to be (TURN) 1000 by a new system of gel-permeation chromatography. A gel permeation column of TSK-G3000 PW that was equilibrated and developed with 36% or 45% acetonitrile in 0.1% trifluoroacetic acid fractionated mixtures of peptides with high resolving power. In addition, the elution volumes of 11 standard peptides and proteins were linearly related to the logarithms of their molecular weights in the acetonitrile-trifluoroacetic acid solvent at both low and high flow rates. Since the solvent is volatile and relatively transparent to short wavelength ultraviolet light this high performance-gel permeation system offers a rapid and highly sensitive method for the analysis, characterization and purification of peptides and proteins from complex mixtures.