Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/9545
Citations | ||
Scopus | Web of ScienceĀ® | Altmetric |
---|---|---|
?
|
?
|
Type: | Journal article |
Title: | The nucleotide-binding site of human sphingosine kinase 1 |
Author: | Pitson, S. Moretti, P. Zebol, J. Zareie, R. Derian, C. Darrow, A. Qi, J. D'Andrea, R. Bagley, C. Vadas, M. Wattenberg, B. |
Citation: | Journal of Biological Chemistry, 2002; 277(51):49545-49553 |
Publisher: | Amer Soc Biochemistry Molecular Biology Inc |
Issue Date: | 2002 |
ISSN: | 0021-9258 1083-351X |
Statement of Responsibility: | Pitson, Stuart M ; Moretti, Paul A B ; Zebol, Julia R ; Zareie, Reza ; Derian, Claudia K ; Darrow, Andrew L ; Qi, Jenson ; D'andrea, Richard J ; Bagley, Christopher J ; Vadas, Mathew A ; Wattenberg, Binks W |
Abstract: | 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 approximately 45-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. |
Keywords: | Cell Line Animals Humans Baculoviridae Azides Phosphotransferases (Alcohol Group Acceptor) Nucleotides Alanine Lysine Calmodulin Recombinant Proteins DNA, Complementary Adenosine Adenosine Triphosphate Affinity Labels Photoaffinity Labels Blotting, Western Transfection Mutagenesis, Site-Directed Gene Deletion Binding Sites Amino Acid Sequence Amino Acid Motifs Catalytic Domain Protein Structure, Tertiary Protein Binding Protein Folding Sequence Homology, Amino Acid Substrate Specificity Kinetics Mutation Catalysis Molecular Sequence Data Mass Spectrometry Insecta |
DOI: | 10.1074/jbc.M206687200 |
Published version: | http://dx.doi.org/10.1074/jbc.m206687200 |
Appears in Collections: | Aurora harvest 4 Medicine publications |
Files in This Item:
There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.