A point mutant of human sphingosine kinase 1 with increased catalytic activity.
Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly(113), a highly conserved residue of human sphingosine kinase 1 (hSK), by site-directed mutagenesis. Surprisingly, a Gly(113)-->Ala substitution generated a mutant that had 1.7-fold greater catalytic activity than wild-type hSK (hSK(WT)). Our data suggests that the Gly(113)-->Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSK(G113A) activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSK(WT).
Pitson
S M
SM
Hanson Centre for Cancer Research, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. stuart.pitson@imvs.sa.gov.au
Moretti
P A
PA
Zebol
J R
JR
Vadas
M A
MA
D'Andrea
R J
RJ
Wattenberg
B W
BW
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D005998:Glycine / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D017510:Protein Folding; D013110:Sphingosine / Q000378:metabolism; D000099157:Sphingosine Kinase
D000409:Alanine; D001224:Aspartic Acid; D005998:Glycine; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase