Acetylation Of Sphingosine Kinase And Its Therapeutic Role In Diabetes Mellitus

Sphingosine kinase1(SPK1) is a key enzyme in sphingolipid metabolism. Weanalyzed the amino acid sequences of SPK1and found that SPK1contained aconserved GK motif that is found in many acetylated proteins, suggesting that SPK1may be acetylated under certain conditions. We determined frstly whether SPK1might be acetylated in vivo. Flag-tagged SPK1(Flag-SPK1) was transfected intoHEK293cells and then cell lysates were immunoprecipitated with Flag M2beads.The immunoprecipitation (IP) products were subjected to Western blot analysis.Acetylated SPK1was detected. For in vitro acetylation assay, the IP products wereincubated with or without p300acetyltransferase in the presence of acetyl-CoA.Incubation with p300acetyltransferase resulted in an increased amount of acetylatedSPK1. Therefore, SPK1could be acetylated both in vivo and in vitro. Next wedetermined whether the two lysine residues in the GK motif of SPK1were acetylated.To do so, the two K in the GK motif of SPK1were mutated either to R or Q, whichwe termed as R-SPK1or Q-SPK1. SPK1acetylation was found only in W-SPK1, butnot in R-SPK1, suggesting that SPK1is acetylated at one or t wo lysines in itsconserved GK motif.This post-translational protein modification of SPK1may be an importantregulation of protein function.Our result showed that acetylation increases SPK1stability. When the two K in the GK motif of SPK1were mutated to R or Q, theubiquitination of SPK1was blocked, which prevented its degradation. These resultsindicated that the processes of acetylation and ubiquitination that involve SPK1compete for the same sites on the two lysine residues in the SPK1GK motif and thatacetylation stabilizes the SPK1protein by block its ubiquitination. Then weinvestigated the effects of SPK1acetyaltion on cell function. The Q-SPK1stablyexpressed cells show a signifcantly larger size when compared with the parentalHEK293cells and with the W-SPK1and R-SPK1stably expressed cells. However,the growth rate of these cells was reduced dramatically. Flow cytometry revealed thatthe proportion of cells in S-phase was increased and the proportion of G2-/M-phasecells was reduced markedly in Q-SPK1stably expressed cells, in comparison to the other cells. Thisfnding indicated that SPK1acetyaltion plays important roles in cellgrowth, cell size and cell cycle progression.Furthermore, we investigated the effects of SPK1in spontaneous KK/Ay type2diabetic mice model using an attenuated salmonella-mediated gene transger approach.It was demonstrated that Q-SPK1could markedly improve blood glucose level andglucose tolerance. Besides, Q-SPK1also lightened body weight and protected againsthepatic and pancreatic organ injury.Also, we investigated the roles of SPK1in skin injury in streptozotocin(STZ)-induced diabetic rat model using plasmid pcDNA3-Flag-SPK1. The resultsshowed that SPK1promoted wound.In conclusion, ourfndings indicated that acetylation regulates SPK1proteinstability, and controls cell growth and cell-cycle progression. And acetylated SPK1can improve blood glucose of type2diabetic mice. And SPK1could significantlyimprove diabetes caused skin damage.