Tài liệu Báo cáo khoa học: Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro

Phosphorylation of hormone-sensitive lipase by protein

kinase A in vitro promotes an increase in its hydrophobic

surface area

Christian Krintel1,2, Matthias Mo¨rgelin3

, Derek T. Logan2 and Cecilia Holm1

1 Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund University, Sweden

2 Department of Molecular Biophysics, Lund University, Sweden

3 Department of Clinical Sciences, Division of Infection Medicine, Lund University, Sweden

Introduction

In mammals, fatty acids are mobilized from stored

triacylglycerols by the consecutive action of adipose

triglyceride lipase (ATGL), hormone-sensitive lipase

(HSL), and monoacylglycerol lipase [1]. Phosphoryla￾tion of HSL by protein kinase A (PKA) is central to

the molecular control of lipolysis, but other events,

notably phosphorylation of the lipid droplet protein

perilipin, are also of key importance. In adipocytes,

stimulation of lipolysis by catecholamines results in

activation of adenylate cyclase, leading to elevated

Keywords

cholesterol ester hydrolase; electron

microscopy; fluorescence spectroscopy;

phospholipid vesicles

Correspondence

C. Holm, Department of Experimental

Medical Science, BMC, C11, SE-221 84

Lund, Sweden

Fax: +46 462224022

Tel: +46 462228581

E-mail: [email protected]

(Received 10 March 2009, revised 17 May

2009, accepted 25 June 2009)

doi:10.1111/j.1742-4658.2009.07172.x

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobili￾zation 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 lm in binding assays. Upon PKA phosphory￾lation, the interactions of HSL with both bis-ANS and the alternative

probe SYPRO Orange were increased. By negative stain transmission elec￾tron microscopy, phosphorylated HSL was found to have a closer interac￾tion 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.

Structured digital abstract

l MINT-7211789: PKA (uniprotkb:P05132) phosphorylates (MI:0217) HSL (uniprotkb:P15304)

by protein kinase assay (MI:0424)

Abbreviations

ATGL, adipose triglyceride lipase; bis-ANS, 4,4¢-dianilino-1,1¢-binaphthyl-5,5¢-disulfonic acid; HSL, hormone-sensitive lipase; LPL, lipoprotein

lipase; PKA, protein kinase A; TO, triolein.

4752 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS