THE INSULIN-DIRECTED PHOSPHORYLATION SITE ON ATP-CITRATE LYASE IS IDENTICAL WITH THE SITE PHOSPHORYLATED BY THE CAMP-DEPENDENT PROTEIN-KINASE INVITRO

Abstract

32P-labeled ATP-citrate lyase isolated form 32P-labeled [rat] hepatocytes treated with insulin contained 1.6- to 1.8-fold greater 32P-radioactivity per mg protein than control enzyme. Both enzyme preparations were digested in parallel with trypsin until 94% of all 32P-radioactivity was rendered acid soluble. Quantitative high performance liquid chromatographic peptide mapping of the tryptic digests revealed a principal 32P-peptide which accounted for at least 80% of the insulin induced increment in 32P-radioactivity of native lyase. This peptide was purified, sequenced and the site of 32P-phosphorylation assigned by 2 methods: electrophoresis (pH 6.5) of residual peptide after each step of Edman degradation and solid phase sequencing. The site of insulin-directed phosphorylation of ATP-citrate lyase (Thr-Ala-Ser(32P)-Phe-Ser-Glu-Ser-Arg) is the same as that directed by glucagon, and, in turn, identical with that phosphorylated by the cAMP-dependent protein kinase in vitro.