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Previous in vitro studies demonstrated that ATP-citrate lyase is phosphorylated by cyclic AMP-dependent protein kinase at peptide A, containing a phosphoserine residue, and by ATP-citrate lyase kinase at peptide B, containing both phosphoserine and phosphothreonine residues (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956). In the present study, trypsin-digested, radiolabeled ATP-citrate lyase from rat epididymal fat pads was analyzed by high performance liquid chromatography. Phosphorylation occurred at three amino acid residues within two different peptide sequences; one (peptide a) contained phosphoserine and the other (peptide b) contained phosphoserine and phosphothreonine. The retention times and molecular weights were the same for peptides a and A and peptides b and B. Isoproterenol action increased peptide a phosphorylation and, to a lesser extent, peptide b phosphorylation. Insulin action also increased peptide a phosphorylation, but did not increase peptide b phosphorylation.

ATP-citrate lyase from rat liver and adipose tissue is phosphorylated by either ATP-citrate lyase kinase or catalytic subunit of cyclic AMP-dependent protein kinase to 0.5-0.6 mol/subunit. We previously demonstrated that the site phosphorylated by ATP-citrate lyase kinase (peptide B) is different from that phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase (peptide A) (Ramakrishna, S., Pucci, D. L., and Benjamin, W.B. (1981) J. Biol. Chem. 256, 10213-10216). ATP-citrate lyase phosphorylation by both protein kinases added simultaneously was increased synergistically. When ATP-citrate lyase was first phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase, the net phosphorylation of the fragments subsequently phosphorylated by lyase kinase increased about 6-fold. However, when ATP-citrate lyase was first phosphorylated by lyase kinase, there was no effect on the subsequent phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Alkaline phosphatase-dephosphorylated ATP-citrate lyase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase to 0.9-1.0 mol/subunit. However, dephospho-ATP-citrate lyase was not phosphorylated by lyase kinase. The addition of both protein kinases simultaneously phosphorylated ATP-citrate lyase up to 2 mol/subunit. Phosphorylation of dephospho-ATP-citrate lyase first by catalytic subunit of cyclic AMP-dependent protein kinase and ATP enabled the lyase to be phosphorylated by lyase kinase. Peptide mapping and phosphoamino acid analysis of dephospho-ATP-citrate lyase phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and/or lyase kinase conclusively showed that phosphorylation of ATP-citrate lyase by ATP-citrate lyase kinase was completely dependent on peptide A phosphorylation by cyclic AMP-dependent protein kinase. Furthermore, increased phosphorylation when both protein kinases were added simultaneously was due to increased phosphorylation at peptide B.