SK1-I is a specific SphK1 inhibitor. (A) Chemical structures of sphingosine, SK1-I (2-amino-3[2-(4-pentylphenyl)ethyl-1-ene]1,3-propanediol), and DMS (N,N-dimethylsphingosine). (B) HEK 293 cells were transiently transfected with vector, V5-hSphK1 or V5-hSphK2. Cell lysates were prepared and equal amounts of proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-V5 antibody. Blots were stripped and reprobed with antiactin as a loading control. (C) SphK1 and SphK2 activities in cell lysates were measured with 10 μM sphingosine in the absence or presence of the indicated concentrations of either SK1-I or 10 μM DMS. SphK1 activity was determined in the presence of Triton X-100 and SphK2 activity was measured in the presence of high salt concentrations, conditions that favor SphK1 and SphK2, respectively. Data are expressed as percentage SphK activity measured in the absence of inhibitor. *P ≤ .01. (D,E) Lineweaver-Burk plots. SphK1 and SphK2 activity was measured with increasing concentrations of sphingosine and the indicated concentrations of SK1-I. Regression analysis revealed a K_m of 10 plus or minus 2 μM, V_max of 38 000 plus or minus 2000 pmol/min per milligram of protein, and K_i of 10 plus or minus 5 μM for SphK1 (D) and K_m of 12 plus or minus 1 μM and V_max 8100 plus or minus 300 pmol/min per milligram of protein for hSphK2 (E), without significant inhibition by SK1-I. (F) Effects of SK1-I on activity of the indicated protein kinases were assessed by SelectScreen Kinase Profiling as described in “Methods.” Data are expressed as percentage of control activity (in the absence of SK1-I) and are averages of 2 independent measurements.