Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

BACKGROUND: 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. CONCLUSIONS: 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

Hormone Sensitive Lipase - in vitro phosphorylation

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

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

Quarternary Structure and Enzymological Properties of the Different Hormone-Sensitive Lipase (HSL) Isoforms

Background: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependent manner, where the predominant 84 kDa form in adipocytes is the most extensively studied. Methodology/Principal Findings: In this study we employed negative stain electron microscopy (EM) to analyze the quarternary structure of the different HSL isoforms. The results show that all three isoforms adopt a head-to-head homodimeric organization, where each monomer contains two structural domains. We also used enzymatic assays to show that despite the variation in the size of the N-terminal domain all three isoforms exhibit similar enzymological properties with regard to psychrotolerance and protein kinase A (PKA)-mediated phosphorylation and activation. Conclusions/Significance: We present the first data on the quaternary structure and domain organization of the three HSL isoforms. We conclude that despite large differences in the size of the N-terminal, non-catalytic domain all three HSL isoforms exhibit the same three-dimensional architecture. Furthermore, the three HSL isoforms are very similar with regar

Immunomodulation—A Molecular Solution to Treating Patients with Severe Bladder Pain Syndrome?

Background: Patients with bladder pain syndrome experience debilitating pain and extreme frequency of urination. Numerous therapeutic approaches have been tested, but as the molecular basis of disease has remained unclear, specific therapies are not available. Objective: Recently, a systematic gene deletion strategy identified interleukin-1 (IL-1) hyperactivation as a cause of severe cystitis in a murine model. Treatment with an IL-1 receptor antagonist (IL-1RA) restored health in genetically susceptible mice, linking IL-1–dependent inflammation to pain and pathology in the bladder mucosa. The study objective was to investigate whether IL-1RA treatment might be beneficial in patients with bladder pain syndrome. Design, setting, and participants: Patients diagnosed with bladder pain syndrome were invited to participate and subjected to daily IL-1RA injections for 1 wk, followed by a treatment break. Patients with other urological disorders accompanied by pain were included as controls. Outcome measurements and statistical analysis: When symptoms returned, treatment was resumed and responding patients were maintained on treatment long term, with individualized dosing regimens. Symptom scores were recorded and molecular effects were quantified by neuropeptide and gene expression analysis. DNA samples were subjected to exome genotyping. Results and limitations: IL-1RA treatment reduced bladder pain and the frequency of urination in 13/17 patients (p < 0.001). Substance P levels in urine were lowered, and responders returned to a more normal lifestyle. Neuroinflammatory-dependent and IL-1–dependent gene networks were inhibited, as well as regulators of innate immunity. Genotyping revealed disease-associated IL1R1, NLRP3, and IL1RN DNA sequence variants in the responders. Controls did not benefit from IL-1RA treatment, except for one patent with cystitis cystica. Conclusions: In this clinical study, IL-1RA treatment is proposed to reduce chronic bladder pain, immediately and in the long term. Despite the limited number of study patients, the potent acute effect and lasting symptom relief indicate that this therapeutic approach may be worth exploring in controlled clinical trials. Patient summary: Treatment with an interleukin-1 (IL-1) receptor antagonist is proposed for treating bladder pain syndrome, as it can result in symptom relief and increase quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects. Take Home Message: Interleukin-1 (IL-1) receptor antagonist immunotherapy is proposed as a new approach to treating bladder pain syndrome, a debilitating disorder. Treated patients experienced symptom relief and increased quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects