Krintel Christian, Osmark Peter, Larsen Martin R, Resjö Svante, Logan Derek T, Holm Cecilia
Division of Diabetes, Metabolism and Endocrinology, Department of Experimental Medical Science, Lund University, Lund, Sweden.
PLoS One. 2008;3(11):e3756. doi: 10.1371/journal.pone.0003756. Epub 2008 Nov 19.
Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo.
METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate.
Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region.
激素敏感脂肪酶（HSL）是从储存的三酰甘油中动员脂肪酸的关键酶。其活性受可逆性蛋白质磷酸化调节。在大鼠中，HSL的Ser563、Ser659和Ser660已被证明在体外和体内均可被蛋白激酶A（PKA）磷酸化。
方法/主要发现：在本研究中，我们采用定点诱变、体外磷酸化和质谱分析表明，PKA对人HSL的体外磷酸化主要发生在Ser649和Ser650（大鼠HSL中的Ser659和Ser660）。野生型酶和四个突变体以C端His标签形式在Sf9昆虫细胞中表达并纯化至均一。Ser552和/或Ser554突变为Ala或Glu的HSL变体保留了脂解和非脂解活性，并被PKA磷酸化，且激活程度与野生型酶相似。（32）P标记研究表明，大部分磷酸化发生在Ser649/Ser650位点，Ser552和Ser554仅有少量磷酸化。串联质谱分析表明，含有Ser649和Ser650的肽主要在Ser650上磷酸化。缺乏所有四个丝氨酸的突变体脂解活性严重降低，但非脂解活性降低程度较小，对丁酸对硝基苯酚酯和三油精的S(0.5)值与野生型HSL相当，且不被PKA磷酸化。野生型酶的PKA磷酸化导致使用TO底物时最大周转率和S(0,5)均增加。
我们的结果表明，PKA在体外主要通过Ser649和Ser650的磷酸化激活人HSL对脂质底物的活性。此外，结果表明Ser649和Ser650位于脂质结合区域附近，且PKA磷酸化控制该区域的可及性。
