Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area.
Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL. Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA. This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate.
Krintel
Christian
C
Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund University, Sweden.
Mörgelin
Matthias
M
Logan
Derek T
DT
Holm
Cecilia
C
D017667:Adipocytes / Q000201:enzymology; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008854:Microscopy, Electron; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000737:chemistry; D002787:Sterol Esterase / Q000737:chemistry* / Q000302:isolation & purification
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D012694:Serine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D017868:Cyclic AMP-Dependent Protein Kinases; D002787:Sterol Esterase