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Originally published In Press as doi:10.1074/jbc.M206687200 on October 18, 2002

J. Biol. Chem., Vol. 277, Issue 51, 49545-49553, December 20, 2002
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The Nucleotide-binding Site of Human Sphingosine Kinase 1*

Stuart M. PitsonDagger §||, Paul A. B. MorettiDagger , Julia R. ZebolDagger , Reza ZareieDagger , Claudia K. Derian**, Andrew L. Darrow**, Jenson Qi**, Richard J. D'AndreaDagger §Dagger Dagger , Christopher J. BagleyDagger §, Mathew A. VadasDagger §§§, and Binks W. WattenbergDagger §§

From the Dagger  Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide SA 5000, Australia, the § Department of Medicine, University of Adelaide, SA 5005, Australia, and ** Johnson & Johnson Pharmaceutical Research & Development, Spring House, Pennsylvania 19477-0776

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 Gly82 of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ~45-fold higher Km(ATP). We have also shown that Lys103 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 SGDGX17-21K 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.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Hanson Institute, Div. of Human Immunology, Inst. of Medical and Veterinary Science, Frome Rd., Adelaide SA 5000, Australia. Tel.: 618-8222-3480; Fax: 618-8232-4092; E-mail: stuart.pitson@imvs.sa.gov.au.

|| Supported by a Georgina Dowling Medical Research Associateship from the University of Adelaide.

Dagger Dagger Supported by an H. M. Lloyd Senior Research Fellowship in Oncology from the University of Adelaide.

§§ Supported by grants from the National Health and Medical Research Council of Australia.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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