Stimulation of tumor cell motility linked to phosphodiesterase catalytic site of autotaxin.
A family of extracellular type I phosphodiesterases has recently been isolated by cDNA cloning, but a physiological function linked to the phosphodiesterase active site has remained unknown. We now present evidence that the phosphodiesterase catalytic site, 201YMRPVYPTKTFPN213, is essential for the motility stimulating activity of autotaxin (ATX), one member of the exophosphodiesterase family. Native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds ATP noncovalently, and undergoes threonine phosphorylation. Homogeneously purified recombinant ATX, based on the teratocarcinoma sequence, retains these same activities. A single amino acid in the phosphodiesterase catalytic site, Thr210, is found to be necessary for motility stimulation, phosphodiesterase activity, and phosphorylation. Two mutant recombinant proteins, Ala210- and Asp210-ATX, lack motility stimulation and lack both enzymatic activities; Ser210-ATX possesses intermediate activities. Another mutation, with the adjacent lysine (Lys209) changed to Leu209-ATX, possesses normal motility stimulation with sustained phosphodiesterase activity but exhibits no detectable phosphorylation. This mutation eliminates the phosphorylation reaction and indicates that the dephosphorylated state is an active motility-stimulating form of the ATX molecule. By demonstrating that the phosphodiesterase enzymatic site is linked to motility stimulation, these data reveal a novel role for this family of exo/ecto-enzymes and open up the possibility of extracellular enzymatic cascades as a regulatory mechanism for cellular motility.
Lee
H Y
HY
Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Clair
T
T
Mulvaney
P T
PT
Woodhouse
E C
EC
Aznavoorian
S
S
Liotta
L A
LA
Stracke
M L
ML
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002465:Cell Movement; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000473:pathology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000099189:Lysophospholipase D
D005956:Glucose-6-Phosphate Isomerase; D006023:Glycoproteins; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D000099189:Lysophospholipase D