We have been studying tumor cell motility as a component of the process of metastasis with a special emphasis on factors that could regulate this motility. To this end, we purified, cloned, and sequenced autotaxin, a tumor-secreted motility-stimulating glycoprotein from the conditioned medium of a human melanoma cell line. Utilizing reverse transciption followed by PCR, we have also isolated autotaxin from human breast carcinoma and teratocarcinoma cell lines. Northern blot analysis of multiple normal tissues indicated that this protein has the highest steady state levels in ovary, small intestine, brain and placenta. In addition to its motility-stimulating properties, autotaxin possesses type I phosphodiesterase, ATPase, nucleotide pyrophosphatase, inorganic pyrophosphatase, 5|-nucleotidase, autophosphorylation, and ATP binding activities. Recombinant autotaxin, purified to homogeneity, retains all of the above activities and stimulates motility at high picomolar to low nanomolar concentrations. Though the human melanoma cell line, A2058, has been used as the motility model in most of the purifications of autotaxin, we have found that certain breast and prostatic carcinoma cells, neuroblastoma cells, bovine aortic smooth muscle cells, as well as HUVEC|s (human umbilical vein cells) also respond to autotaxin as a chemoattractant. Studies utilizing site-directed mutagenesis of the phosphodiesterase active site demonstrated that an intact phosphodiesterase catalytic site is necessary for motility, but phosphorylation of the molecule does not appear to be a prerequisite. Since autotaxin has no known ATP binding site, we utilized a radiolabelled 8-azido-ATP to demonstrate ATP binding to autotaxin and to localize two regions on the molecule where the 8-azido-ATP crosslinks after exposure to light. The first region is the phosphodiesterase active site itself. The second region is 108 amino acids downstream, is unlike any reported ATP binding sites, and is sensitive to addition of salt. We have found that autotaxin possesses type I phosphodiesterase, ATPase, nucleotide pyrophosphatase, autophosphorylation, and ATP binding activities. Utilizing site- directed mutagenesis, we explored the relationship between the enzymatic properties of autotaxin and its capacity to stimulate motility. We found that a single point mutation in the phosphodiesterase catalytic site (Thr210 Ala or Asp) inhibited its phosphodiesterase, autophosphorylation, and motility-stimlating activities but had no effect on ATP binding. A second point mutation (Lys209 Leu) inhibited autophosphorylation but not motility stimulation or phosphodiesterase. Therefore, an intact phosphodiesterase catalytic site is necessary for motility, but phosphorylation of the molecule does not appear to be a prerequisite. Finally, we have found that AMP and adenosine are chemoattractants for the human melanoma cell line, A2058. These molecules, which are products of autotaxin's interaction with ATP, appear to act through an A1 adenosine receptor. Transfection of autotaxin into 3T3 cells which have an activated ras oncogene profoundly stimulates the invasive, tumorigenic and metastatic properties of these cells, compared to controls.Further studies have shown that the induction of invasion and metastasis is augmented by a stimulation of angiogenesis.
| Clair, Timothy; Aoki, Junken; Koh, Eunjin et al. (2003) Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate. Cancer Res 63:5446-53 |
| Woodhouse, Elisa C; Fisher, Amy; Bandle, Russell W et al. (2003) Drosophila screening model for metastasis: Semaphorin 5c is required for l(2)gl cancer phenotype. Proc Natl Acad Sci U S A 100:11463-8 |
