Autotaxin Is an Exoenzyme Possessing 5′-Nucleotide Phosphodiesterase/ATP Pyrophosphatase and ATPase Activities

Abstract

Autotaxin (ATX)1 is a 125-kDa glycoprotein secreted by the human melanoma cell line A2058. ATX stimulates both random and directed motility in its producer cells (1), and its recent cloning and sequencing (2) has revealed homology with the active site of bovine intestinal 5Ј-nucleotide PDE (EC 3.1.4.1) (4) and extensive homology with the ectoprotein PC-1 (5), the brain-type PDE I-nucleotide pyrophosphatase gene 2 (6), and the rat neural differentiation antigen gp130 RB13-6 (7). ATX contains two tandem somatomedin B regions, the loop region of an EF-hand and a type I PDE catalytic site, and possesses 5Ј-nucleotide PDE activity (2) .Early studies on digestive enzymes responsible for RNA degradation identified a class of enzymes characterized by their reaction product, a 5Ј-monophosphate nucleotide, and their activity toward p-nitrophenyl-thymidine monophosphate (⌽-TMP) (8). This type I PDE activity has also been detected in a variety of mammalian tissues, their plasma membranes, and cell surfaces (9 -11). The unifying features of these activities, in addition to the reaction product, are the broad specificity for substrates and competitive inhibitors, the alkaline pH optimum, and the ability to hydrolyze the phosphodiester bond between the ␣-and ␤-phosphates in nucleoside polyphosphates. ATX possesses type I PDE activity and also induces a known biological response, the potent stimulation of cellular locomotion; thus it is possible to investigate the role of this enzyme reaction center in extracellular signal transduction.The reaction mechanism for type I PDE has been described as involving formation of nucleotidylated threonine as a covalently bound reaction intermediate (4), and PC-1 can be autophosphorylated on this threonine at the PDE catalytic center using [␥-32 P]ATP (12). Previous studies from this laboratory on ATX with point mutations at the PDE active site showed that the corresponding threonine in ATX (Thr 210 ) is required for its chemotactic, 5Ј-nucleotide PDE and threonine phosphorylation activities, and that phosphorylation-deficient, 5Ј-nucleotide PDE-competent ATX (K209L) is fully active in the stimulation of cellular motility (3). These findings suggested that the dephosphorylated state of ATX is a biologically active form and prompted us to investigate the relationship between the phosphorylation state and the catalytic properties of ATX. These earlier studies had also shown that phospho-ATX contains the ␥-and not the ␣-phosphate from ATP but addressed neither the stability of this construct nor the fate of the ␤-phosphate. In addition, unanswered questions remained concerning the nucleotide reaction products, the ability of ATX to use substrates other than ATP, and the possibility that the phosphorylation of ATX was due to the presence of a co-purifying protein kinase. We have resolved these issues by characterizing the enzymatic activities of ATX using homogeneously pure recombinant ATX (rATX) derived from the human teratocarcinoma cell line N-tera2D1 (13) and partially purified ATX (A2058 AT...

Cellular function and molecular structure of ecto-nucleotidases

“…This result corroborates earlier observations that NPP2 cleaves PP i into P i [364]. This would implicate a considerable overlap in the catalytic properties of TNAP and NPPs.…”

“…Mutation of these three glycines into alanine abolished the nucleotide phosphodiesterase activity of NPP1 [366]. Yet, recombinant NPP2 prepared and isolated from a vaccinia virus lysate of BS-C-1 cells was found to hydrolyze both ATP [364] and dinucleoside polyphosphates [351].…”

NAD+ and ATP Released from Injured Cells Induce P2X7-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells

“…Enzymes capable of reversing ADP-ribosylation include ADP-ribosylhydrolases which can remove the entire ADP-ribose moiety (72,73) and phosphodiesterases which remove only AMP, leaving ribose phosphate attached to the target protein (74). To date ADP-ribosylhydrolases have been described only as intracellular proteins (75), whereas phosphodiesterase isoforms have been cloned and characterized that function as membrane bound and secretory ecto-enzymes (76,77). Because both labels used in this study (etheno-adenosine) and (␣-32 P) would be removed by phosphodiesterases and ADP-ribosylhydrolases, other tools will be required to determine the relative contributions of these two enzyme families to reversion of protein ADP-ribosylation.…”

Microarray analysis identifies Autotaxin, a tumour cell motility and angiogenic factor with lysophospholipase D activity, as a specific target of cell transformation by v-Jun

“…Owing to this dramatic induction, and because ATX modulates a variety of cellular processes potentially relevant to oncogenesis, we decided to investigate the relationship between v-Jun transformation and ATX expression and activity in more detail. ATX is an ecto-phosphodiesterase/lysophospholipase D (Clair et al, 1997;Umezu-Goto et al, 2002), which is expressed on the cell surface and can be released into the medium via proteolysis (Bollen et al, 2000). Western blotting of cell extracts using an ATX-specific antibody Tables 1 and 2 revealed that v-Jun-transformed CEF expressed readily detectable ATX (ckATX), whereas control CEF did not ( Figure 3a).…”