Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly 82 of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ϳ45-fold higher K m(ATP) . We have also shown that Lys 103 is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX 17-21 K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.

This article is available online at http://www.jlr.org Triglycerides (TGs) are the chief route of transport of dietary fat within chylomicrons and VLDLs, as well as the main form of fuel storage in adipose tissue. TGs are synthesized from one glycerol and three FA molecules, which are attached via ester bonds to the hydroxyl groups of the glycerol backbone. Two major diacylglycerol acyltransferase (DGAT) isozymes, DGAT1 and DGAT2, have been identifi ed. Although both enzymes convert diacylglycerol to TG, they do not share similarity in either their nucleotide or amino acid sequences and have most probably arisen by convergent evolution ( 1, 2 ). Although there are some differences in their tissue distributions, both DGAT1 and DGAT2 are highly expressed in organs that synthesize large amounts of TG, such as the liver, adipose tissue, and small intestine ( 3 ).Studies with genetically altered mice, as well as in vivo suppression of DGAT expression, indicate that both DGAT1 and DGAT2 play important roles in TG synthesis. DGAT1 knockout mice (DGAT1 Ϫ / Ϫ ) have reduced tissue TG levels and exhibit increased sensitivity to insulin and leptin ( 4 ). In addition, they are resistant to high-fat dietinduced obesity as a result of an increase in their metabolic rates ( 4 ). In contrast, knockout mice lacking DGAT2 (DGAT2 Ϫ / Ϫ ) are lipopenic and die soon after birth as a result of profound reductions in substrates for energy metabolism and impaired skin permeability ( 5 ). Hepatic suppression of DGAT2 with antisense oligonucleotides (ASOs) reduced hepatic TG content in rodents ( 6, 7 ), and reversed diet-induced hepatic steatosis and insulin resistance Abstract Diacylglycerol acyltransferase (DGAT) catalyzes the fi nal step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with

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Recent studies have indicated that urotensin II (UII), a cyclic peptide, is vasoactive and may be involved in cardiovascular dysfunctions. It remains unknown, however, whether UII plays a role in the control of renal vascular tone and tubular function. In the present study, a continuous infusion of synthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodium excretion (UV and UNaV) in a dose-dependent manner. At a dose of 20 ng. kg-1. min-1, it increased RBF by 20% and UV and UNaV by 94 and 109%, respectively. Nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) completely abolished hUII-induced increases in RBF and water/sodium excretion. In isolated, pressurized, and phenylephrine-precontracted small RA with internal diameter of approximately 200 microm, hUII produced a concentration-dependent vasodilation with a maximal response of 55% at 1.5 microM. l-NAME significantly blocked this hUII-induced vasodilation by 60%. In denuded RA, hUII had neither vasodilator nor vasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-based fluorescence imaging analysis of NO levels, hUII (1 microM) was shown to double the NO levels within the endothelium of freshly dissected small RA, and l-NAME blocked this UII-induced production of endothelial NO. These results indicate that UII produces vasodilator and natriuretic effects in the kidney and that UII-induced vasodilation is associated with increased endothelial NO in the RA.

We have discovered two related chemical series of nonpeptide urotensin-II (U-II) receptor antagonists based on piperazino-phthalimide (5 and 6) and piperazino-isoindolinone (7) scaffolds. These structure types are distinctive from those of U-II receptor antagonist series reported in the literature. Antagonist 7a exhibited single-digit nanomolar potency in rat and human cell-based functional assays, as well as strong binding to the human U-II receptor. In advanced pharmacological testing, 7a blocked the effects of U-II in vitro in a rat aortic ring assay and in vivo in a rat ear-flush model. A discussion of U-II receptor antagonist pharmacophores is presented, and a specifically defined model is suggested from tricycle 13, which has a high degree of conformational constraint.