Activation and phosphorylation adipose tissue hormone-sensitive lipase by cyclic AMP-dependent protein kinase

Abstract

1. 1. Hormone-sensitive lipase purified approximately 100-fold from rat adipose tissue was activated 50 to 100% by incubation with cyclic AMP, ATP-Mg 2+ and a protein kinase preparation from rabbit muscle. Activation of this purified lipase depended totally on addition of protein kinase and both cyclic AMP and ATP. The apparent K m for cyclic AMP was 1.1 · 10 −7 M; that for ATP was 5 · 10 −6 M. 2. 2. In the presence of [ψ- 32P]ATP, activation was accompanied by transfer of radioactivity to lipase and the bound radioactivity showed properties compatible with those of serine O-phosphate. The time course for phosphorylation closely paralleled that for lipase activation. The apparent K m for cyclic AMP in the phosphorylation reaction was the same as that for activation within experimental error. No phosphorylation was observed in the absence of cyclic AMP, but there was a small but significant degree of phosphorylation not dependent on added protein kinase. 3. 3. In cruder fractions full activation was obtained on addition of cyclic AMP and ATP-Mg 2+ alone, presumably reflecting the presence of sufficient endogenous protein kinase in these fractions. 4. 4. When tissue had been exposed to epinephrine before homogenization neither the cruder fractions nor the purified lipase could be activated. 5. 5. These results strongly support the conclusion that hormonal activation of adipose tissue lipase is mediated by cyclic AMP, that the cyclic AMP acts via protein kinase, and that the conversion of inactive lipase is linked to enzyme phosphorylation. The possibility that the high-molecular-weight lipase preparation includes other enzymes, specifically a “lipase kinase” analogous to Krebs' phosphorylase b kinase, cannot be ruled out.