Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution

Sphingosine-1-phosphate (SPP), the product of sphingosine kinase, is an imp ortant signaling molecule with intra- and extracellular functions. The cDNA for the mouse sphingosine kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human sphi ngosine kinase (huSPHK1). Sequence analysis comparison revealed that this k inase is evolutionarily very conserved, having a high degree of homology wi th the murine enzyme, and presenting several conserved regions with bacteri a, yeast, plant, and mammalian proteins. Expressed huSPHK1 cDNA specificall y phosphorylates D-erythro-sphingosine and, to a lesser extent, D,L-erythro -dihydrosphingosine, and not at all the 'threo' isoforms of dihydrosphingos ine; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosph atidylinositol (PI); phosphatidylinositol-4-phosphate (PIP); or phosphatidy linositol-4,5-bisphosphate (PIP2). huSPHK1 shows typical Michaelis-Menten k inetics (V-max = 56 mu M and K-m = 5 mu M). The kinase is inhibited by D,L- threo-dihydrosphingosine (K-i = 3 mu M), and by N,N-dimethyl-sphingosine (K -i = 5 mu M). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respecti vely. It is also expressed in brain and heart. In addition, database search es with the stSG2854 sequence indicate that huSPHK1 is also expressed in en dothelial cells, retinal pigment epithelium, and senescent fibroblasts. (C) 2000 Elsevier Science B.V. All rights reserved.