Hormone Sensitive Lipase - in vitro phosphorylation

Abstract

Abstract Fatty acids mobilized from triacylglycerol stores are a major energy source in humans. Mobilization occurs through the consecutive action of three lipases: adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). Whereas ATGL and MGL only hydrolyse tri- and monoacylglycerols, respectively, HSL has a broad substrate range and hydrolyses cholesterol esters and soluble esters as well as mono-, di- and triacylglycerols. Unlike other lipases the 84 kDa adipocyte isoform of HSL comprises, apart from the catalytic domain, a 36 kDa N-terminal domain involved in protein-protein interactions and a 150 amino acid regulatory module, neither of which show any homology to other proteins. In addition to the 84 kDa isoform two larger isoforms of 117 kDa and 89 kDa are expressed in testis and β-cells respectively. These isoforms are identical to the adipocyte isoform apart from N-terminal extensions, which could be speculated to form individual globular domains. A major feature of HSL is its regulation by reversible phosphorylation. Previous work has shown that HSL is phosphorylated by cAMP activated protein kinase A (PKA) on Ser563, Ser659 and Ser660 (rat HSL numbering) in vitro. Phosphorylation by PKA results in increased HSL activity in vitro as well as in vivo. However, whereas in vitro activation has only been seen against triglycerides and is usually in the range of 1 to 2-fold, catecholamine stimulation of adipocyte can increase lipolysis up to 100-fold. The results presented in this thesis show that the major PKA site in vitro is Ser650 in human HSL (corresponding to Ser660 in rat HSL) and that phosphorylation by PKA confers increases in Km and Vmax against triolein. It is also shown that PKA phosphorylation increases activity in vitro towards cholesterol esters, diglycerides and triglycerides. Using rat HSL we show that in vitro HSL activation by PKA is caused by an increase in hydrophobic surface area upon phosphorylation. Moreover we demonstrate that the three different HSL isoforms are phosphorylated and activated equally by PKA in vitro. The three isoforms show similar relative lipase activities, but differ in their activities towards soluble esters. Furthermore, electron microscopy reveals that HSL is a homodimer and that this organization is conserved in all three isoforms without the N-terminal extensions of the testis and β-cell isoforms forming separate domains