An insulin-sensitive cytosolic protein kinase accounts for the regulation of ATP citrate-lyase phosphorylation

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Abstract
Purified rat liver ATP citrate-lyase is phosphorylated on serine residues by an insulin-stimulated cytosolic kinase activity partially purified from rat adipocytes [Yu, Khalaf and Czech (1987) J. Biol. Chem. 262, 16677-16685). The Km for lyase phosphorylation by this hormone-sensitive kinase activity is approx. 3 .mu.M. Two-dimensional tryptic-peptide mapping of the 32P-labelled lyase reveals that the kinase-catalysed phosphorylation occurs primarily on a specific peptide. In intact 32P-labelled adipocytes, insulin enhances the serine phosphorylation of ATP citrate-lyase by 2-3-fold. Tryptic digestion of the 32P-labelled lyase immunopurified from insulin-treated adipocytes also yields one major phosphopeptide. 32P-labelled lyase tryptic peptides derived from labelling experiments in vitro and in vivo exhibit identical electrophoretic and chromatographic migration profiles. Furthermore, radio-sequencing of the phosphopeptide from lyase 32P-labelled in vitro indicates that serine-3 from the N-terminus is phosphorylated by the insulin-stimulated cytosolic kinase, in agreement with previous studies on the position of the phosphoserine residue in ATP citrate-lyase isolated from insulin-treated cells. Taken together, the similarity in site-specific phosphorylation of ATP citrate-lyase from insulin-treated adipocytes to that catalysed by the hormone-activated cytosolic kinase in vitro strongly suggests that this kinase mediates insulin action on lyase phosphorylation in intact cell.