Dimethylsphingosine and FTY720 inhibit the SK1 form but activate the SK2 form of sphingosine kinase from rat heart.
Fractionation of cytosolic sphingosine kinase (SKase) activity by gel filtration chromatography gave rise to a 96-kDa peak that contained only the SK2 form of SKase (by Western analysis) and a broad ca. 46 kDa peak that contained only SK1 forms. SK2 appeared to have a bound accessory protein. When tested with the classic SKase inhibitor dimethylsphingosine (DMS), SK1 was extensively inhibited; however, SK2 was not inhibited but unexpectedly was activated. Activation of SK2 was the result of DMS enhancing the affinity of the enzyme for sphingosine, and, at low concentrations of ATP and sphingosine, activated by more than 100%. Activation of SK2 could be demonstrated in the cytosolic fraction indicating it was unrelated to the purification step. The immunomodulator FTY720 also activated SK2 (although to a lesser extent), but was a potent inhibitor of SK1. SK2 from rat liver and spleen was also not inhibited by DMS. L-Sphingosine and to a lesser extent dihydrosphingosine and phytosphingosine were effective inhibitors of both forms.
Vessey
Donald A
DA
Liver Study Unit, Department of Veterans Affairs Medical Center, San Francisco, CA 94121, USA. donald.vessey@med.va.gov
Kelley
Michael
M
Zhang
Jianqing
J
Li
Luyi
L
Tao
Rong
R
Karliner
Joel S
JS
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D000068876:Fingolimod Hydrochloride; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011409:Propylene Glycols / Q000494:pharmacology*; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000099157:Sphingosine Kinase
D000906:Antibodies; D000068876:Fingolimod Hydrochloride; D007527:Isoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); D011409:Propylene Glycols; D013110:Sphingosine; D000099157:Sphingosine Kinase; C061800:N,N-dimethylsphingosine