Asp177 in C4 domain of mouse sphingosine kinase 1a is important for the sphingosine recognition

• Published in: FEBS letters Vol. 578; no. 1; pp. 106 - 110
• Main Authors: Yokota, Shinji, Taniguchi, Yuki, Kihara, Akio, Mitsutake, Susumu, Igarashi, Yasuyuki
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 03.12.2004
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Aspartic Acid - metabolism; Calmodulin; Calmodulin - metabolism; calmodulin/Sepharose 4B; CaM-Sepharose; Cell Line; ceramide kinase; CERK; DGK; diacylglycerol kinase; Humans; Isoenzymes - genetics; Isoenzymes - metabolism; Mice; Molecular Sequence Data; mouse sphingosine kinase 1a; mSK1a; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein Binding; Protein Folding; Protein Structure, Tertiary; S1P; Sequence Alignment; Sph; Sphingosine; Sphingosine - metabolism; Sphingosine 1-phosphate; Sphingosine kinase; TBS; Tris-buffered saline; Sphingosine kinase; S1P; mSK1a; CERK; TBS; Sphingosine; SK; DGK; Sph; CaM-Sepharose; Calmodulin; Sphingosine 1-phosphate
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2004.10.081

Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp 177 → Asn 177, caused a dramatic decrease in SK activity (to ∼6% of wild type) and an increase in the K m value for Sph (10.1 → 108 μM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp 177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK.