Autotaxin Is an Exoenzyme Possessing 5′-Nucleotide Phosphodiesterase/ATP Pyrophosphatase and ATPase Activities
1997; Elsevier BV; Volume: 272; Issue: 2 Linguagem: Inglês
10.1074/jbc.272.2.996
ISSN1083-351X
AutoresTimothy Clair, Hoi Young Lee, Lance A. Liotta, Mary L. Stracke,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoAutotaxin (ATX) is an extracellular enzyme and an autocrine motility factor that stimulates pertussis toxin-sensitive chemotaxis in human melanoma cells at picomolar to nanomolar concentrations. This 125-kDa glycoprotein contains a peptide sequence identified as the catalytic site in type I alkaline phosphodiesterases (PDEs), and it possesses 5′-nucleotide PDE (EC 3.1.4.1) activity (Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Årestad, A., Cioce, V., Schiffmann, E., and Liotta, L. (1992) J. Biol. Chem. 267, 2524-2529; Murata, J., Lee, H. Y., Clair, T., Krutsch, H. C., Årestad, A. A., Sobel, M. E., Liotta, L. A., and Stracke, M. L. (1994) J. Biol. Chem. 269, 30479-30484). ATX binds ATP and is phosphorylated only on threonine. Thr210 at the PDE active site of ATX is required for phosphorylation, 5′-nucleotide PDE, and motility-stimulating activities (Lee, H. Y., Clair, T., Mulvaney, P. T., Woodhouse, E. C., Aznavoorian, S., Liotta, L. A., and Stracke, M. L. (1996) J. Biol. Chem. 271, 24408-24412). In this article we report that the phosphorylation of ATX is a transient event, being stable at 0°C but unstable at 37°C, and that ATX has adenosine-5′-triphosphatase (ATPase; EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Thus ATX catalyzes the hydrolysis of the phosphodiester bond on either side of the β-phosphate of ATP. ATX also catalyzes the hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and the hydrolysis of NAD to AMP, and each of these substrates can serve as a phosphate donor in the phosphorylation of ATX. ATX possesses no detectable protein kinase activity toward histone, myelin basic protein, or casein. These results lead to the proposal that ATX is capable of at least two alternative reaction mechanisms, threonine (T-type) ATPase and 5′-nucleotide PDE/ATP pyrophosphatase, with a common site (Thr210) for the formation of covalently bound reaction intermediates threonine phosphate and threonine adenylate, respectively. Autotaxin (ATX) is an extracellular enzyme and an autocrine motility factor that stimulates pertussis toxin-sensitive chemotaxis in human melanoma cells at picomolar to nanomolar concentrations. This 125-kDa glycoprotein contains a peptide sequence identified as the catalytic site in type I alkaline phosphodiesterases (PDEs), and it possesses 5′-nucleotide PDE (EC 3.1.4.1) activity (Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Årestad, A., Cioce, V., Schiffmann, E., and Liotta, L. (1992) J. Biol. Chem. 267, 2524-2529; Murata, J., Lee, H. Y., Clair, T., Krutsch, H. C., Årestad, A. A., Sobel, M. E., Liotta, L. A., and Stracke, M. L. (1994) J. Biol. Chem. 269, 30479-30484). ATX binds ATP and is phosphorylated only on threonine. Thr210 at the PDE active site of ATX is required for phosphorylation, 5′-nucleotide PDE, and motility-stimulating activities (Lee, H. Y., Clair, T., Mulvaney, P. T., Woodhouse, E. C., Aznavoorian, S., Liotta, L. A., and Stracke, M. L. (1996) J. Biol. Chem. 271, 24408-24412). In this article we report that the phosphorylation of ATX is a transient event, being stable at 0°C but unstable at 37°C, and that ATX has adenosine-5′-triphosphatase (ATPase; EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Thus ATX catalyzes the hydrolysis of the phosphodiester bond on either side of the β-phosphate of ATP. ATX also catalyzes the hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and the hydrolysis of NAD to AMP, and each of these substrates can serve as a phosphate donor in the phosphorylation of ATX. ATX possesses no detectable protein kinase activity toward histone, myelin basic protein, or casein. These results lead to the proposal that ATX is capable of at least two alternative reaction mechanisms, threonine (T-type) ATPase and 5′-nucleotide PDE/ATP pyrophosphatase, with a common site (Thr210) for the formation of covalently bound reaction intermediates threonine phosphate and threonine adenylate, respectively. INTRODUCTIONAutotaxin (ATX) 1The abbreviations used are: ATXautotaxinrATXrecombinant ATXPDEphosphodiesteraseϕ-TMPp-nitrophenyl-thymidine monophosphateAMP-CPadenosine 5′-(α,β-methylenediphosphate)AMP-PCPadenosine 5′-(β,γ-methylenetriphosphateAMP-CPPadenosine 5′-(α,β-methylenetriphosphate)PAGEpolyacrylamide gel electrophoresis. is a 125-kDa glycoprotein secreted by the human melanoma cell line A2058. ATX stimulates both random and directed motility in its producer cells (1Stracke M.L. Krutzsch H.C. Unsworth E.J. Årestad A. Cioce V. Schiffmann E. Liotta L. J. Biol. Chem. 1992; 267: 2524-2529Google Scholar), and its recent cloning and sequencing (2Murata J. Lee H.Y. Clair T. Krutsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Google Scholar) has revealed homology with the active site of bovine intestinal 5′-nucleotide PDE (EC 3.1.4.1) (4Culp J.S. Blytt H.J. Hermodson M. Butler L.G. J. Biol. Chem. 1985; 260: 8320-8324Google Scholar) and extensive homology with the ectoprotein PC-1 (5Buckley M.F. Loveland K.A. McKinstry W.J. Garson O.M. Goding J.W. J. Biol. Chem. 1990; 265: 17506-17511Google Scholar), the brain-type PDE I-nucleotide pyrophosphatase gene 2 (6Kawagoe H. Soma O. Goji J. Nishimura N. Narita M. Inazawa J. Nakamura H. Sano K. Genomics. 1995; 30: 380-384Google Scholar), and the rat neural differentiation antigen gp130RB13-6 (7Deissler H. Lottspeich F. Rajewsky M.F. J. Biol. Chem. 1995; 270: 9849-9855Google Scholar). ATX contains two tandem somatomedin B regions, the loop region of an EF-hand and a type I PDE catalytic site, and possesses 5′-nucleotide PDE activity (2Murata J. Lee H.Y. Clair T. Krutsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Google Scholar).Early studies on digestive enzymes responsible for RNA degradation identified a class of enzymes characterized by their reaction product, a 5′-monophosphate nucleotide, and their activity toward p-nitrophenyl-thymidine monophosphate (Φ-TMP) (8Razzell W.E. Khorana H.G. J. Biol. Chem. 1959; 234: 2105-2113Google Scholar). This type I PDE activity has also been detected in a variety of mammalian tissues, their plasma membranes, and cell surfaces (9Decker K. Bischoff E. FEBS Lett. 1971; 21: 95-98Google Scholar, 10Evans W.H. Hood D.O. Gurd J.W. Biochem. J. 1973; 135: 819-826Google Scholar, 11Morley D.J. Hawley D.M. Ulbright T.M. Butler L.G. Culp J.S. Hodes M.E. J. Histochem. Cytochem. 1987; 35: 75-82Google Scholar). 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 α- and β-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 (4Culp J.S. Blytt H.J. Hermodson M. Butler L.G. J. Biol. Chem. 1985; 260: 8320-8324Google Scholar), and PC-1 can be autophosphorylated on this threonine at the PDE catalytic center using [γ-32P]ATP (12Oda Y. Kuo M.-D. Huang S.S. Huang J.S. J. Biol. Chem. 1993; 268: 27318-27326Google Scholar). Previous studies from this laboratory on ATX with point mutations at the PDE active site showed that the corresponding threonine in ATX (Thr210) is required for its chemotactic, 5′-nucleotide PDE and threonine phosphorylation activities, and that phosphorylation-deficient, 5′-nucleotide PDE-competent ATX (K209L) is fully active in the stimulation of cellular motility (3Lee H.Y. Clair T. Mulvaney P.T. Woodhouse E.C. Aznavoorian S. Liotta L.A. Stracke M.L. J. Biol. Chem. 1996; 271: 24408-24412Google Scholar). 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 γ- and not the α-phosphate from ATP but addressed neither the stability of this construct nor the fate of the β-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 (13Lee H.Y. Murata J. Clair T. Polymeropoulos M.H. Torres R. Manrow R. Liotta L.A. Stracke M.L. Biochem. Biophys. Res. Commun. 1996; 218: 714-719Google Scholar) and partially purified ATX (A2058 ATX) from A2058 human melanoma cells.DISCUSSIONIn this study we have shown that homogeneously pure rATX catalyzes 5′-nucleotide PDE activity (Fig. 1) under physiological conditions and is indistinguishable from A2058 ATX (purified from a human melanoma cell line), based on the kinetics of threonine phosphorylation and dephosphorylation (Fig. 2, Fig. 3). In addition we have shown that, with ATP as a substrate, ATX has ATPase (producing ADP and Pi) and ATP pyrophosphatase (producing AMP and PPi) activities (Fig. 4).Since both the 5′-nucleotide PDE and ATP pyrophosphatase activities of ATX hydrolyze the α-β phosphodiester bond in their respective nucleotide substrates, it is probable that these two activities result from the same reaction mechanism. ATX is labeled by either [32P](adenylate) NAD or [32P]AMP (this report) but not by [α-32P] ATP (3; data not shown). It is possible that ATX preferentially hydrolyzes, and incorporates phosphate from, the highest energy phosphoester bond in the substrate, which, in the case of ATP, is the β-γ phosphodiester bond. Such a preference would also explain the observations that ATP and ϕ-TMP are comparable in their ability to compete for the noncovalent binding of [α-32P]8-N3-ATP to ATX (Fig. 5A), but that ATP is effective at much lower concentrations than ϕ-TMP in inhibiting phosphorylation of ATX by [γ-32P]ATP (Fig. 5B). Consistent with this possibility is the observation (Fig. 5C) that ATP derivatives that lack a hydrolyzable bond at the β-γ position (AMP-CP and AMP-PCP) are less effective as inhibitors of the ATX-catalyzed 5′-nucleotide PDE reaction than derivatives that contain a hydrolyzable bond at this position (AMP-CPP and ATP). The interesting suggestion (17Uriarte M. Stalmans W. Hickman S. Bollen M. Biochem. J. 1995; 306: 271-277Google Scholar) that there may be competition between the phosphorylation and phosphodiesterase activities of PC-1 may be relevant to these unresolved questions regarding ATX. The simplest interpretation of the competition between substrates for ATP binding, 5′-nucleotide PDE, and phosphorylation (Fig. 5) is that a single nucleotide binding site is used by ATX for each of these enzymatic functions, but definitive resolution of this question awaits more extensive enzyme inhibition and nucleotide binding studies.GTP, NAD, AMP, and PPi are susceptible to hydrolysis by ATX and serve as phosphate donors in its phosphorylation. The hydrolysis of PPi to Pi occurs in a number of intracellular energy-conserving reactions (18Reeves R.E. Trends Biochem. Sci. 1976; 1: 53-55Google Scholar), but the relationship between these reactions and the inorganic pyrophosphatase activity of ATX is not clear. The predominant products of ATP hydrolysis by ATX in vitro are ADP and Pi, but the substrates and products of in vivo catalysis by ATX in the stimulation of tumor cell motility are not known. With the ability to hydrolyze nucleoside polyphosphates at a variety of positions, ATX may catalyze nucleotidase cascades (19Pearson J.D. Carleton J.S. Gordon J.L. Biochem. J. 1980; 190: 421-429Google Scholar, 20Trams E.G. J. Theor. Biol. 1980; 87: 609-621Google Scholar). ATX hydrolyzes substrates other than ATP, and the facility with which these substrates phosphorylate ATX suggests that in catalyzing each of the various hydrolytic reactions, ATX uses a covalently bound, phosphate-containing reaction intermediate. The data presented in this article strongly suggest that this is indeed the case for the ATPase reaction catalyzed by ATX. Fig. 6 depicts a proposed model for the formation of covalently bound reaction intermediates in the catalytic action of ATX toward ATP. ATX is proposed to be capable of at least two alternative mechanisms, ATPase and 5′-nucleotide PDE/ATP pyrophosphatase, each of which uses Thr210 as the site for the formation of the covalently bound reaction intermediate. The phosphothreonine intermediate in the ATPase reaction mechanism (Fig. 6, reaction 1) contains only the γ-phosphate from ATP and is stable at 0°C and unstable at 37°C (Fig. 2, Fig. 3). The depicted formation of the adenylyl threonine intermediate (Fig. 6, reaction 2) is based on the reported mechanism for 5′-nucleotide PDE (4Culp J.S. Blytt H.J. Hermodson M. Butler L.G. J. Biol. Chem. 1985; 260: 8320-8324Google Scholar). According to this proposal the phosphorylation-dephosphorylation cycle of ATX is a integral part of the ATPase reaction mechanism, and ATX is atypical among known ATPases (21Pedersen P.L. Carafoli E. Trends Biochem. Sci. 1987; 12: 146-150Google Scholar, 22Plesner L. Int. Rev. Cytol. 1995; 158: 141-214Google Scholar) in that it uses a phosphorylated threonine as a covalently bound reaction intermediate. Unequivocal demonstration of the identity of the phosphorylation-dephosphorylation cycle of ATX with its ATPase activity awaits analysis in progress designed to show that a single point mutation simultaneously abolishes both of these activities. This mutational analysis is also being used to investigate the possibility that the same relationship holds between the other phosphorylation substrates and their hydrolysis by ATX.Among the proteins with sequence homology to ATX the most well characterized is the ectoprotein PC-1. ATX and PC-1 each contain two tandem somatomedin B regions, the loop region of an EF-hand, and a type I PDE catalytic site and possess 5′-nucleotide PDE activity (2Murata J. Lee H.Y. Clair T. Krutsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Google Scholar, 23Rebbe N.F. Tong B.D. Finley E.M. Hickman S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5192-5196Google Scholar). Studies on the effect of pH on the PDE activity of PC-1 (12Oda Y. Kuo M.-D. Huang S.S. Huang J.S. J. Biol. Chem. 1993; 268: 27318-27326Google Scholar), assayed at a substrate (ϕ-TMP) concentration of 0.5 mM, show optimum activity at alkaline pH, a characteristic that is typical of type I PDE enzymes. The 5′-nucleotide PDE activity of ATX at submillimolar substrate concentrations (Fig. 1) does not show this preference for alkaline pH. These data suggest that ATX and PC-1 may differ in this respect, and that catalysis of the 5′-nucleotide PDE reaction by ATX is physiologically relevant. [α-32P]ATP has been reported to label purified PC-1 (threonine at the PDE active site) (12Oda Y. Kuo M.-D. Huang S.S. Huang J.S. J. Biol. Chem. 1993; 268: 27318-27326Google Scholar) as well as immunoprecipitated or cell surface PC-1 (24Belli S.I. Mercuri F.A. Sali A. Goding J.W. Eur. J. Biochem. 1995; 228: 669-676Google Scholar). Attempts to label ATX with [α-32P]ATP have been unsuccessful (3; data not shown). It is possible that adenylyl ATX, formed during incubation of ATP with ATX, exists only as a short-lived 5′-nucleotide PDE/ATP pyrophosphatase reaction intermediate and that its extremely transient nature precludes detection under the conditions and quantities of ATX used. Such a characteristic would also explain the efficiency of this ATX-catalyzed reaction at physiological pH, a property previously unreported among the type I PDE enzymes. The dephosphorylation of phospho-ATX also differs from that of PC-1 in that it occurs after dialysis to remove exogenous nucleotides, which are reported to be stimulatory and necessary for the dephosphorylation of phospho-PC-1 (17Uriarte M. Stalmans W. Hickman S. Bollen M. Biochem. J. 1995; 306: 271-277Google Scholar).This and other distinctions in the enzymatic characteristics between PC-1 and ATX may arise, at least in part, from a difference in the sequence of the nucleotide binding site. PC-1 (5Buckley M.F. Loveland K.A. McKinstry W.J. Garson O.M. Goding J.W. J. Biol. Chem. 1990; 265: 17506-17511Google Scholar) contains the glycine-rich GXGXXG sequence found in nucleotide-binding proteins (25Schulz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Google Scholar) along with the downstream lysine invariably found in protein kinases (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Google Scholar), and this region may serve as an ATP binding site. Although ATX (2Murata J. Lee H.Y. Clair T. Krutsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Google Scholar) has extensive homology to PC-1, it does not contain this sequence, nor does ATX contain a perfect match to any of the other P-loop type sequences found in adenine and guanine nucleotide-binding proteins (27Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Google Scholar). Although the nature of the ATP binding site(s) in PDE enzymes is not yet defined, ATP clearly binds to ATX (3; this report), and both PC-1 (28Maddux B.A. Sbraccia P. Kumakura S. Sasson S. Youngren J. Fisher A. Spencer S. Grupe A. Henzel W. Stewart T.A. Reaven G.M. Goldfine I.D. Nature. 1995; 373: 448-451Google Scholar) and ATX (3; this report) have been purified to homogeneity using ATP-agarose chromatography. The failure to detect protein kinase activity in ATX is not unexpected considering the lack of sequence similarity to known protein kinases.The discovery of the heterotrimeric G-proteins and the nature of their interaction with adenylyl cyclase (29Gilman A. Biosci. Rep. 1995; 15: 65-97Google Scholar) revealed a mechanism by which intracellular enzyme catalysis participates in signal transduction, but such a role for extracellular enzyme activity has not been established. Human angiogenin has been reported to have both RNase and angiogenic activities (30Shapiro R. Biochemistry. 1986; 25: 3527-3532Google Scholar); the thymidine phosphorylase activity of platelet-derived endothelial cell growth factor may be responsible for its chemotactic activity (31Haraguchi M. Miyadera K. Uemura K. Sumizawa T. Furukawa T. Yamada K. Akiyama S. Nature. 1994; 368: 198Google Scholar); antibodies directed against alkaline phosphatase activity have been shown to inhibit cell migration during development of the axolotl pronephric duct (32Drawbridge J. Scherson T. Erdman J.E. Basaviah P. Steinberg M.S. J. Cell Biol. 1991; 115: 145aGoogle Scholar); and the pertussis toxin-sensitive stimulation of tumor cell motility by ATX requires an intact 5′-nucleotide PDE reaction site (3Lee H.Y. Clair T. Mulvaney P.T. Woodhouse E.C. Aznavoorian S. Liotta L.A. Stracke M.L. J. Biol. Chem. 1996; 271: 24408-24412Google Scholar). Together these observations suggest that extracellular enzyme catalysis may also have a role in transmembrane signaling.Extracellular nucleosides and nucleotides participate in a variety of biological processes, including signal transduction through purinoreceptors (33Stiles G. J. Biol. Chem. 1992; 267: 6451-6454Google Scholar, 34Chen Z.-P. Levy A. Lightman S.L. J. of Neuroendocrinol. 1995; 7: 83-96Google Scholar) and nucleoside phosphate and phosphoprotein metabolism by ectoenzymes (35vanDriel I.R. Wilks A.F. Pietersz G.A. Goding J.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8619-8623Google Scholar, 36Lin S.-H. J. Biol. Chem. 1989; 264: 14403-14407Google Scholar, 37Ehrlich Y.H. Hogan M.V. Pawlowska Z. Naik U. Kornecki E. Ann. N. Y. Acad. Sci. 1990; 603: 401-416Google Scholar, 38Najjar S.M. Accili D. Phillipe N. Jernberg J. Margolis R. Taylor S.I. J. Biol. Chem. 1993; 268: 1201-1206Google Scholar, 39Wang T.-F. Guidotti G. J. Biol. Chem. 1996; 271: 9898-9901Google Scholar, 40Strobel R.S. Nagy A.K. Knowles A.F. Buegel J. Rosenberg M.D. J. Biol. Chem. 1996; 271: 16323-16331Google Scholar). Adenosine has been shown to promote angiogenesis in the chick egg system (41Dusseau J.W. Hutchins P.M. Respir. Physiol. 1988; 71: 33-44Google Scholar) and chemotaxis in endothelial (42Meininger C.J. Schelling M.E. Granger H.J. Am. J. Physiol. 1988; 255: H554-H562Google Scholar) and immune (43Rose F.R. Hirschhorn R. Weissman G. Cronstein B.N. J. Exp. Med. 1988; 167: 1186-1194Google Scholar) cells and to have complex effects on chemotaxis in neutrophils (44Garcia-Castro I. Mato J. Vasanthakumar G. Weismann W. Schiffmann E. Chiang P. J. Biol. Chem. 1983; 258: 4345-4349Google Scholar). Extracellular nucleotidases such as ATX may serve to deplete ATP and/or ADP as a cytoprotective mechanism (45Suprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Google Scholar) or to terminate P2 purinoreceptor-mediated signals (34Chen Z.-P. Levy A. Lightman S.L. J. of Neuroendocrinol. 1995; 7: 83-96Google Scholar). Also, enzyme catalysis by ATX may provide AMP and/or adenosine to initiate P1 purinoreceptor-mediated signals (33Stiles G. J. Biol. Chem. 1992; 267: 6451-6454Google Scholar), or it may participate in salvage pathways by facilitating the capture and reuptake of nucleosides (46Che M. Nishida T. Gatmaitan Z. Arias I.M. J. Biol. Chem. 1992; 267: 9684-9688Google Scholar). Cell adhesion molecule 105 has been identified as an ecto-ATPase with implications for cell-cell interaction (47Aurivillius M. Hansen O.C. Lazrek M.B.S. Bock E. Öbrink B. FEBS Lett. 1990; 264: 267-269Google Scholar), and a rat liver ecto-ATPase has been identified as a canalicular bile acid transport protein (48Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Google Scholar). Since phosphorylation-deficient ATX (K209L) is biologically active (3Lee H.Y. Clair T. Mulvaney P.T. Woodhouse E.C. Aznavoorian S. Liotta L.A. Stracke M.L. J. Biol. Chem. 1996; 271: 24408-24412Google Scholar), the ATPase activity of ATX may be dispensable for the stimulation of cellular motility. On the other hand, the stability of phospho-ATX in vivo is unknown, and since the dephospho form of ATX is apparently an active state, the possibility of a regulatory role for the phosphorylation of ATX is not excluded.Continuing investigations on autotaxin are designed to test the hypothesis that the phosphorylated forms of ATX are enzyme-bound reaction intermediates in the hydrolysis of phosphoester bonds and to study the relationship between the 5′-nucleotide PDE/ATP pyrophosphatase activity of ATX and its stimulation of cellular motility, as well as the influence of the phosphorylation state and ATPase activities on these properties of ATX. INTRODUCTIONAutotaxin (ATX) 1The abbreviations used are: ATXautotaxinrATXrecombinant ATXPDEphosphodiesteraseϕ-TMPp-nitrophenyl-thymidine monophosphateAMP-CPadenosine 5′-(α,β-methylenediphosphate)AMP-PCPadenosine 5′-(β,γ-methylenetriphosphateAMP-CPPadenosine 5′-(α,β-methylenetriphosphate)PAGEpolyacrylamide gel electrophoresis. is a 125-kDa glycoprotein secreted by the human melanoma cell line A2058. ATX stimulates both random and directed motility in its producer cells (1Stracke M.L. Krutzsch H.C. Unsworth E.J. Årestad A. Cioce V. Schiffmann E. Liotta L. J. Biol. Chem. 1992; 267: 2524-2529Google Scholar), and its recent cloning and sequencing (2Murata J. Lee H.Y. Clair T. Krutsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Google Scholar) has revealed homology with the active site of bovine intestinal 5′-nucleotide PDE (EC 3.1.4.1) (4Culp J.S. Blytt H.J. Hermodson M. Butler L.G. J. Biol. Chem. 1985; 260: 8320-8324Google Scholar) and extensive homology with the ectoprotein PC-1 (5Buckley M.F. Loveland K.A. McKinstry W.J. Garson O.M. Goding J.W. J. Biol. Chem. 1990; 265: 17506-17511Google Scholar), the brain-type PDE I-nucleotide pyrophosphatase gene 2 (6Kawagoe H. Soma O. Goji J. Nishimura N. Narita M. Inazawa J. Nakamura H. Sano K. Genomics. 1995; 30: 380-384Google Scholar), and the rat neural differentiation antigen gp130RB13-6 (7Deissler H. Lottspeich F. Rajewsky M.F. J. Biol. Chem. 1995; 270: 9849-9855Google Scholar). ATX contains two tandem somatomedin B regions, the loop region of an EF-hand and a type I PDE catalytic site, and possesses 5′-nucleotide PDE activity (2Murata J. Lee H.Y. Clair T. Krutsch H.C. Årestad A.A. Sobel M.E. Liotta L.A. Stracke M.L. J. Biol. Chem. 1994; 269: 30479-30484Google Scholar).Early studies on digestive enzymes responsible for RNA degradation identified a class of enzymes characterized by their reaction product, a 5′-monophosphate nucleotide, and their activity toward p-nitrophenyl-thymidine monophosphate (Φ-TMP) (8Razzell W.E. Khorana H.G. J. Biol. Chem. 1959; 234: 2105-2113Google Scholar). This type I PDE activity has also been detected in a variety of mammalian tissues, their plasma membranes, and cell surfaces (9Decker K. Bischoff E. FEBS Lett. 1971; 21: 95-98Google Scholar, 10Evans W.H. Hood D.O. Gurd J.W. Biochem. J. 1973; 135: 819-826Google Scholar, 11Morley D.J. Hawley D.M. Ulbright T.M. Butler L.G. Culp J.S. Hodes M.E. J. Histochem. Cytochem. 1987; 35: 75-82Google Scholar). 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 α- and β-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 (4Culp J.S. Blytt H.J. Hermodson M. Butler L.G. J. Biol. Chem. 1985; 260: 8320-8324Google Scholar), and PC-1 can be autophosphorylated on this threonine at the PDE catalytic center using [γ-32P]ATP (12Oda Y. Kuo M.-D. Huang S.S. Huang J.S. J. Biol. Chem. 1993; 268: 27318-27326Google Scholar). Previous studies from this laboratory on ATX with point mutations at the PDE active site showed that the corresponding threonine in ATX (Thr210) is required for its chemotactic, 5′-nucleotide PDE and threonine phosphorylation activities, and that phosphorylation-deficient, 5′-nucleotide PDE-competent ATX (K209L) is fully active in the stimulation of cellular motility (3Lee H.Y. Clair T. Mulvaney P.T. Woodhouse E.C. Aznavoorian S. Liotta L.A. Stracke M.L. J. Biol. Chem. 1996; 271: 24408-24412Google Scholar). 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 γ- and not the α-phosphate from ATP but addressed neither the stability of this construct nor the fate of the β-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 (13Lee H.Y. Murata J. Clair T. Polymeropoulos M.H. Torres R. Manrow R. Liotta L.A. Stracke M.L. Biochem. Biophys. Res. Commun. 1996; 218: 714-719Google Scholar) and partially purified ATX (A2058 ATX) from A2058 human melanoma cells.
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