Phosphorylation of the Immunomodulatory Drug FTY720 by Sphingosine Kinases

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, SPHK2 isoform was more than SPHK2 kinase and a sphingosine and and 2 of and 2 in murine of SPHK2 sphingosine was lower than of SPHK1, FTY720 phosphorylation was SPHK2. human while of sphingosine was than of FTY720 phosphorylation was SPHK2 FTY720 was phosphorylated in human as in the of and in of SPHK2 in human and 2 of SPHK2 is more than The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, SPHK2 isoform was more than SPHK2 kinase and a sphingosine and and 2 of and 2 in murine of SPHK2 sphingosine was lower than of SPHK1, FTY720 phosphorylation was SPHK2. human while of sphingosine was than of FTY720 phosphorylation was SPHK2 FTY720 was phosphorylated in human as in the of and in of SPHK2 in human and 2 of SPHK2 is more than FTY720 is immunomodulatory is in of and of was found in in FTY720 a resulting the of the of FTY720 for the effects of the drug FTY720 phosphate as of the receptors for sphingosine-1-phosphate sphingosine sphingosine of sphingosine sphingosine of is is the by FTY720 FTY720 was found in FTY720 phosphate as the is responsible for of of the been phosphorylated by and of and is phosphorylated in of FTY720 the of the of the FTY720 was a for murine sphingosine kinase 1a of FTY720 the and of phosphorylation of phosphorylation of was for receptors in and as the was in in was phosphorylation of FTY720 by type is for biological a isoform been and the and human type 2 been human type 2 in the and the a for and been for the of was in and and of SPHK2 in and because been as a and in and a the and 2 been and of and been the phosphorylation of FTY720 by and 2 in and by the in murine and in human We that, while FTY720 phosphorylation in vivo, SPHK2 is the more in the FTY720 the of and 2 in the in and was and was FTY720 and and the and of and in and in human and in 2 in of and was by human and and the was the is a of the the of the was the in in and the the and the and the the for and for and as The and in by the was and and The was by the was the The was and the and in and the as as was in and in and the and was also by in and for kinase was in a of was a human and the and The was by of the in the and and the for of the of was and and in was also by and in of and 2 of murine and a for as for The of the was the and 2 in and of the for the the of and of for of and SPHK2. was in a as a by and by as of the of of in in of kinase phosphate for and and of and in and the was in for the of the 2 as in was in as the of in a and of the of was by of in was in of sphingosine FTY720 as a as for sphingosine in of and 2 of as in and and as of and in of by of the of and 2 and the of the was in the of and the was in for for and by of in of and of and of the was and in The was in and a as the and FTY720 phosphate and a and by of was for the was SPHK2 in the of and of was as for the The was found in the of the of sphingosine and FTY720 as in the of phosphorylated the by the of was as of of the the of in was as by as the of in by a and was the of the the for and The for was The of and of the of a was for the and as in a of as by of of as 2 of and of by for and of in the was in the for in the of of the of for a The was the of the the as the as the the of the of and a a the by was the is as the in a of and as the for of and in a of in in was in by and was also The and in in of phosphate and for and the was in of the as for was was found in the of the is as of phosphorylated of of in of and 2 in was as for the was 2 of in was human and The was of and in the as sphingosine as the and the phosphorylation of FTY720 was was of and for of the was of and in in of and as for the by and by by The of the phosphate was the of the FTY720 and FTY720 phosphate of and We the human sphingosine type and 2 in The as in of the was the and in and 2 of the was and of of the the of and 2 a of of for of the of sphingosine and a the Km and Km for sphingosine for and while the was for the of and 2 in the was a of the of the of the the of is than of sphingosine as of and 2 in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in a FTY720 the in of of the and as by FTY720 phosphate in and FTY720 as the of of the FTY720 and Km of the compound. the Km and in as the the Km for FTY720 was and the lower than for the of the Km for FTY720 was of the of as the for while was lower the of and 2 phosphorylates 30-fold more efficiently, the lower Km of FTY720 for was also the of a of FTY720 biological a for the human in the of phosphorylation of was than is a also FTY720 than The of was phosphorylated by also the murine sphingosine 1a and 2 in and sphingosine and FTY720 as The Km and and 2 been the of the human and murine and for and phosphorylation of FTY720 by the type 2 the phosphorylates more than in the human and and been We a for splice variants in of the human and found variants of the by the and and found of the is by the variants in and sphingosine and the in and the of variants the of sphingosine was as of a of phosphorylation of FTY720 a in the of the the of the by a human and for the Among the by a kinase for of SPHKs, and in a as The and kinase and a The a of a the of the of lower than the for the as of is and and and and and a and a the of The of the is a while of the is and in the as is by a of The of is the of a and the of a was as by and the as by and and sphingosine FTY720 the and in of a kinase was in as sphingosine the in by and also the of kinase as as in the human and 2 and splice variants sphingosine and of and 2 and in the far, and 2 the FTY720 and SPHK2 is more efficient in FTY720 than We the also in vivo, the and of and 2 in found the for was in of in and by and and in by was in by and is in by of in by of in by for and 2 as as and the in and 2 in the We the is by while SPHK2 is by the and 2 The of in was by in while of was by in in for the the of in the of the and 2 of and 2 of phosphorylation of sphingosine was in and 2 2 and phosphorylation of sphingosine and FTY720 in of by the of was in the of in and as phosphorylation of FTY720 and in of human and was for and the phosphate was in the was the of and a and a by was also and the of and the for and sphingosine FTY720 as as of phosphorylated and of sphingosine was as was in the the and the in and and in the The in and in the for in as of and was in the was found in the while in and was a of the of in of of of of as of of of of FTY720 as in a SPHK2 was lower than by of in while in and SPHK2 was than SPHK2 was in the in and and in is in the SPHK2 the of and the SPHK2 the SPHK2 of SPHK2 SPHK2 was found in the the of and the was a the was phosphorylated more in the SPHK2 of as SPHK2 sphingosine in was lower than SPHK2 FTY720 and sphingosine was in and and 2 of FTY720 in of FTY720 phosphate SPHK2 more in the as a lower SPHK1, is in the the more efficient phosphorylation of FTY720 by the of in the found the in the also in in had and 2 in in the and also in and the in the of of FTY720 in of the FTY720 in human the of as a in sphingosine was as SPHK2 while FTY720 phosphorylation was lower by a of in FTY720 is phosphorylated by SPHK2 is more of FTY720 in of the of phosphorylation of FTY720 in of human and by FTY720 the in the was more in and than in human by the in human as and SPHK2 in human was as for FTY720 of is a and of of is of a of and the FTY720 phosphate is in the human is and and FTY720 in vivo, while the in the human as the of the of in human and in the of and 2 the the and the the in and by the of and as the of SPHK2 is more efficient FTY720 than is by the of FTY720 phosphorylation in in human and in human SPHK2 The SPHK2 is the responsible for FTY720 phosphate in is of for of FTY720 also the phosphate of for FTY720 and the of the the FTY720 is immunomodulatory is in of and of was found in in FTY720 a resulting the of the of FTY720 for the effects of the drug FTY720 phosphate as of the receptors for sphingosine-1-phosphate sphingosine sphingosine of sphingosine sphingosine of is is the by FTY720 FTY720 was found in FTY720 phosphate as the is responsible for of of the sphingosine-1-phosphate kinase kinase human sphingosine kinase human murine sphingosine kinase of sphingosine kinase sphingosine-1-phosphate kinase kinase human sphingosine kinase human murine sphingosine kinase of sphingosine kinase FTY720 been phosphorylated by and of and is phosphorylated in of FTY720 the of the of the FTY720 was a for murine sphingosine kinase 1a of FTY720 the and of phosphorylation of phosphorylation of was for receptors in and as the was in in was phosphorylation of FTY720 by type is for biological a isoform been and the and human type 2 been human type 2 in the and the a for and been for the of was in and and of SPHK2 in and because been as a and in and a the and 2 been and of and been the phosphorylation of FTY720 by and 2 in and by the in murine and in human We that, while FTY720 phosphorylation in vivo, SPHK2 is the more in the FTY720 the of and 2 in the in and was and was FTY720 and and the and of and in and in human and in 2 in of and was by human and and the was the is a of the the of the was the in in and the the and the and the the for and for and as The and in by the was and and The was by the was the The was and the and in and the as as was in and in and the and was also by in and for kinase was in a of was a human and the and The was by of the in the and and the for of the of was and and in was also by and in of and 2 of murine and a for as for The of the was the and 2 in and of the for the the of and of for of and SPHK2. was in a as a by and by as of the of of in in of kinase phosphate for and and of and in and the was in for the of the 2 as in was in as the of in a and of the of was by of in was in of sphingosine FTY720 as a as for sphingosine in of and 2 of as in and and as of and in of by of the of and 2 and the of the was in the of and the was in for for and by of in of and of and of the was and in The was in and a as the and FTY720 phosphate and a and by of was for the was SPHK2 in the of and of was as for the The was found in the of the of sphingosine and FTY720 as in the of phosphorylated the by the of was as of of the the of in was as by as the of in by a and was the of the the for and The for was The of and of the of a was for the and as in a of as by of of as 2 of and of by for and of in the was in the for in the of of the of for a The was the of the the as the as the the of the of and a a the by was the is as the in a of and as the for of and in a of in in was in by and was also The and in in of phosphate and for and the was in of the as for was was found in the of the is as of phosphorylated of of in of and 2 in was as for the was 2 of in was human and The was of and in the as sphingosine as the and the phosphorylation of FTY720 was was of and for of the was of and in in of and as for the by and by by The of the phosphate was the of the FTY720 and FTY720 phosphate and was and was FTY720 and and the and of and in and in human and in 2 in of and was by human and and the was the is a of the the of the was the in in and the the and the and the the for and for and as The and in by the SPHK2 was and and The was by the was the The was and the and in and the as as was in and in and the and was also by in and for kinase was in a of was a human and the and The was by of the in the and and the for of the of was and and in was also by and in of and 2 of murine and a for as for The of the was the and 2 in and of the for the the of and of for of and SPHK2. was in a as a by and by as of the of of in in of kinase phosphate for and and of and in and the was in for the of the 2 as in was in as the of in a and of the of was by of in was in of sphingosine FTY720 as a as for sphingosine in of and 2 of as in and and as of and in of by of the of and 2 and the of the was in the of and the was in for for and by of in of and of and of the was and in The was in and a as the and FTY720 phosphate and a and by of was for the was SPHK2 in the of and of was as for the The was found in the of the of sphingosine and FTY720 as in the of phosphorylated the by the of was as of of the the of in was as by as the of in by a and was the of the the for and The for was The of and of the of a was for the and as in a of as by of of as 2 of and of by for and of in the was in the for in the of of the of for a The was the of the the as the as the the of the of and a a the by was the is as the in a of and as the of in in was in by and was also The and in in of phosphate and for and the was in of the as for was was found in the of the is as of phosphorylated of of in of and 2 in was as for the was 2 of in was human and The was of and in the as sphingosine as the and the phosphorylation of FTY720 was was of and for of the was of and in in of and as for the by and by by The of the phosphate was the of the FTY720 and FTY720 phosphate of and We the human sphingosine type and 2 in The as in of the was the and in and 2 of the was and of of the the of and 2 a of of for of the of sphingosine and a the Km and Km for sphingosine for and while the was for the of and 2 in the was a of the of the of the the of is than of sphingosine as of and 2 in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in because of the the was in a FTY720 the in of of the and as by FTY720 phosphate in and FTY720 as the of of the FTY720 and Km of the compound. the Km and in as the the Km for FTY720 was and the lower than for the of the Km for FTY720 was of the of as the for while was lower the of and 2 phosphorylates 30-fold more efficiently, the lower Km of FTY720 for was also the of a of FTY720 biological a for the human in the of phosphorylation of was than is a also FTY720 than The of was phosphorylated by also the murine sphingosine 1a and 2 in and sphingosine and FTY720 as The Km and and 2 been the of the human and murine and for and phosphorylation of FTY720 by the type 2 the phosphorylates more than in the human and and been We a for splice variants in of the human and found variants of the by the and and found of the is by the variants in and sphingosine and the in and the of variants the of sphingosine was as of a of phosphorylation of FTY720 a in the of the the of the by a human and for the Among the by a kinase for of SPHKs, and in a as The and kinase and a The a of a the of the of lower than the for the as of is and and and and and a and a the of The of the is a while of the is and in the as is by a of The of is the of a and the of a was as by and the as by and and sphingosine FTY720 the and in of a kinase was in as sphingosine the in by and also the of kinase as as in the human and 2 and splice variants sphingosine and of and 2 and in the far, and 2 the FTY720 and SPHK2 is more efficient in FTY720 than We the also in vivo, the and of and 2 in found the for was in of in and by and and in by was in by and is in by and 2 in the We the is by while SPHK2 is by the and 2 The of in was by in while of was by in in for the the of in the of the and 2 of and 2 of phosphorylation of sphingosine was in and 2 2 and phosphorylation of sphingosine and FTY720 in of by the of was in the of in and as phosphorylation of FTY720 and in of human and was for and the phosphate was in the was the of and a and a by was also and the of and the for and sphingosine FTY720 as as of phosphorylated and of sphingosine was as was in the the and the in and and in the The in and in the for in as of and was in the was found in the while in and was a of the of in of of of of as of of of of FTY720 as in a SPHK2 was lower than by of in while in and SPHK2 was than SPHK2 was in the in and and in is in the SPHK2 the of and the SPHK2 the SPHK2 of SPHK2 SPHK2 was found in the the of and the was a the was phosphorylated more in the SPHK2 of as SPHK2 sphingosine in was lower than SPHK2 FTY720 and sphingosine was in and and 2 of FTY720 in of FTY720 phosphate SPHK2 more in the as a lower SPHK1, is in the the more efficient phosphorylation of FTY720 by the of in the found the in the also in in had and 2 in in the and also in and the in the of of FTY720 in of the FTY720 in human the of as a in sphingosine was as SPHK2 while FTY720 phosphorylation was lower by a of in FTY720 is phosphorylated by SPHK2 is more of FTY720 in of the of phosphorylation of FTY720 in of human and by FTY720 the in the was more in and than in human by the in human as and SPHK2 in human was as for FTY720 of of and We the human sphingosine type and 2 in The as in of the was the and in and 2 of the was and of of the the of and 2 a of of for of the of sphingosine and a the Km and Km for sphingosine for and while the was for the of and 2 in the was a of the of the of the the of is than of sphingosine as FTY720 the in of of the and as by FTY720 phosphate in and FTY720 as the of of the FTY720 and Km of the compound. the Km and in as the the Km for FTY720 was and the lower than for the of the Km for FTY720 was of the of as the for while was lower the of and 2 phosphorylates 30-fold more efficiently, the lower Km of FTY720 for We was also the of a of FTY720 biological a for the human in the of phosphorylation of was than is a also FTY720 than The of was phosphorylated by We also the murine sphingosine 1a and 2 in and sphingosine and FTY720 as The Km and and 2 been the of the human and murine and for and phosphorylation of FTY720 by the type 2 the phosphorylates more than in the human and and been We a for splice variants in of the human and found variants of the by the and and found of the is by the We variants in and sphingosine and the in and the of variants the of sphingosine was as of a of phosphorylation of FTY720 a in the of the the of the by a We human and for the Among the by a kinase for of SPHKs, and in a as The and kinase and a The a of a the of the of lower than the for the The as of is and and and and and a and a the of The of the is a while of the is and in the as is by a of The of is the of a and the of a was as by and the as by and and sphingosine FTY720 the and in of a kinase was in as sphingosine the in by and also the of kinase as as in the human and 2 and splice variants sphingosine and of and 2 and in the far, and 2 the FTY720 and SPHK2 is more efficient in FTY720 than We the also in vivo, the and of and 2 in found the for was in of in and by and and in by was in by and is in by and 2 in the We the is by while SPHK2 is by the and 2 The of in was by in while of was by in in for the the of in the of the and 2 We of and a and a by was also and the of and the for and sphingosine FTY720 as as of phosphorylated and of sphingosine was as was in the the and the in and and in the The in and in the for in as of and was in the was found in the while in and was a of the SPHK2 was lower than by of in while in and SPHK2 was than SPHK2 was in the in and and in is in the SPHK2 the of and the SPHK2 the SPHK2 of SPHK2 SPHK2 was found in the the of and the was a the FTY720 was phosphorylated more in the SPHK2 of as SPHK2 sphingosine in was lower than SPHK2 FTY720 and sphingosine was in and and 2 of FTY720 in of FTY720 phosphate SPHK2 more in the as a lower SPHK1, is in the the more efficient phosphorylation of FTY720 by SPHK2. the of in the found the in the also in in had and 2 in in the and also in and the in the of of FTY720 in of the FTY720 in human the of as a in sphingosine was as SPHK2 while FTY720 phosphorylation was lower by a of in FTY720 is phosphorylated by SPHK2 is more of FTY720 in of the of phosphorylation of FTY720 in of human and by FTY720 the in the was more in and than in human by the in human as and SPHK2 in human was as for FTY720 of is a and of of is of a of and the FTY720 phosphate is in the human is and and FTY720 in vivo, while the in the human as the of the of in human and in the of and 2 the the and the the in and by the of and as the of SPHK2 is more efficient FTY720 than is by the of FTY720 phosphorylation in in human and in human SPHK2 The SPHK2 is the responsible for FTY720 phosphate in is of for of FTY720 also the phosphate of for FTY720 and the of the the FTY720 is a and of of is of a of and the FTY720 phosphate is in the human is