THEJOURNAL OF BIOLOGICALCHEMISTRY Vol. 269, No. 48, Issue of December 2, pp.

3047930484, 1994
0 1994 by T h e American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

cDNA Cloningof the Human Tumor Motility-stimulating Protein,

Autotaxin, Reveals a Homology with Phosphodiesterases*
(Received for publication, August 2, 1994, and in revised form, September 16, 1994)

Jun Murata, Hoi Young Lee, Timothy Clair, HenryC. Krutzsch, AndersA. Lestad,
Mark E. Sobel, LanceA. Liotta, and Mary L. StrackeS
From the Laboratory of Pathology, National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland 20892

A human cDNAclone encoding autotaxin, a tumor cell This motile response is abolished by pretreatment of t h e cells
is with pertussis toxin. ATX may therefore act througha G pro-
motility-stimulating protein, reveals that this protein
anecto/exo-enzymewithsignificanthomologytothe tein-linked cell surface receptor.
plasma cell membrane differentiation antigen PC-1.ATX Sequence information, obtained initially on 19 purified tryp-
is a 125-kDaglycoprotein,previouslyisolatedfrom a tic peptides, confirmed that the protein is unique with no sig-
human melanomacell line(A2058),which elicits chemo- nificant homology to growth factors or previously described
tactic and chemokinetic responses at picomolar to nano- motility factors. These peptide sequences have now been used
molar concentrations. Affinity-purified antipeptide an- as the basis for identifyingand sequencing thecDNA clone for
tibodies to theATX peptide, ATX-102, were employed to ATX.
screen anA2058 cDNA expression library made in Agtll. EXPERIMENTALPROCEDURES
ThepartialcDNAsequencewhichwasobtainedwas
Materials-Mi-Gel 10 affinity resin was from Bio-Rad. The Gene-
then extendedby utilizing reverse transcriptase on total Amp PCR Reagent kit with AmpliTaq and the GeneAmp RNA PCRkit
cellular RNAfollowed by polymerase chain reaction am- were purchased from Perkin-Elmer. The 5’ RACE kit was fromLife
plificatiop. The isolated cDNAclone contained 3251 base Technologies, Inc.The p-nitrophenyl thymidine-5’-monophosphate was
pairs, and the mRNA message size was approximately obtained from Calbiochem Biochemicals.
3.3 kilobases. The deduced amino acid sequence of au- Cell Culture-The human melanoma cell line A2058, originally iso-
totaxin matched 30 previously sequenced peptides and lated by Todaro et al. (5),was maintained as described previously (6).
The N-tera 2 (Dl clone) was a kind gift from Dr. Maxine Singer, Labo-
comprised a protein of 915 amino acids. Data base anal- ratory of Biochemistry, National Cancer Institute, National Institutes
ysis of theATX sequence revealed a 45% amino acid iden- of Health, and was maintained as described (7).
tity(including 30 outof33 cysteines)withPC-1,a Purification of Autotaxin-The purification ofATX has beende-
pyrophosphatase/type I phosphodiesteraseexpressed scribed previouslyin detail (3) and involved successivefractionations of
on the surface ATX, A2058 serum-free conditioned medium using phenyl-Sepharose CL-4B
of activated B cells and plasma cells.
like PC-1, was found to hydrolyze the type I phosphodi- (Pharmacia Biotech Inc.), agarose-bound C o d (Vector Laboratories
esterasesubstratep-nitrophenylthymidine-5‘-mono- Inc.), ZORBAXBioSeries-WAX (weak anion exchange, MacMod),
Spherogel-TSK 4000SW and 3000SW (TosoHaas), and Pro-Pac PA1
phosphate. Autotaxin now defines a novel motility-regu-(strong anion exchange, Dionex Corp.)chromatographic separations. In
lating function for this class of ecto/exo-enzymes. some cases, chromatographic fractionation with ZORBAX BioSeries
WCX (weak cation exchange, Mac Mod) was utilized between the weak
anion exchange and the gel filtration steps. For this chromatographic
Active tumor cell motility is involved in many stagesof t h e procedure, the motility-stimulating peak fromthe weak anion exchange
metastatic cascade, including the transition from in situ to column was pooled and dialyzed into 25 mM Bistris, 20%(v/v) ethylene
glycol (pH 6.5). Proteins were eluted with a linear gradient of sodium
invasive carcinoma(1). The regulation of this motile responseis
chloride (0-0.3 M).
not well understood. However, multiple factors, both autocrine Production of Anti-ATXPeptide Antibodies-Anti-ATX peptide anti-
and paracrine in origin, appear to influence this tumor cell bodies were generated as described previouslywith slight modification
locomotion (2). (8, 9). In brief, the peptide termed ATX-102 (3) was synthesized on a
Recently, a potent new cytokine with molecular mass 125 Biosearch 9600 peptide synthesizer and conjugated to bovine serum
kDa has been purified to homogeneity from the conditioned albumin using glutaraldehyde. For the first injection into New Zealand
medium of the human melanoma cell line, A2058, utilizing White rabbits, the bovine serum albumin-peptide conjugate was emul-
sified with complete Freund’s adjuvant and injected subcutaneously.
sequential chromatographic methods (3). This new cytokine,
-
For subsequent injections, the bovine serum albumin-peptide conjugate
termed autotaxin (ATX),l is a basic glycoprotein with PI 7.8. was emulsified with incomplete Freund’sadjuvant. The resultant anti-
ATX is active in the high picomolar to low nanomolar range, serum was heat-inactivated at 56 “C for 30min. Immunoglobulins were
stimulating both chemotactic and chemokinetic responses in precipitated in 47% saturated ammonium sulfate, then redissolved and
the ATX-producing A2058 cells as well as other tumor cells(4). dialyzed into phosphate-bufferedsaline. Antibodies wereadsorbed onto
peptide-conjugatedAffi-Gel 10 resin, eluted with 0.1 N acetic acid, and
neutralized with 2 M Tris-HC1 (pH 8). The resulting affinity-purified
* The costs of publication of this article were defrayed in part by the antibodies weredialyzed into Dulbecco’s phosphate-buffered saline,
payment of page charges. This article must therefore be hereby marked concentrated, and stored in aliquots at -20 “C.
“aduertisement” in accordance with 18 U.S.C. Section 1734 solely to Gel EZectrophoresis-Protein samples were analyzed by SDS-polyac-
indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted rylamide gel electrophoresis in a Tris glycine buffersystem as described
to the G’enBankmIEMBLData Bank with accession number(s) L35594. by Laemmli (lo), using prepared 8-16% gradient gels (Novex).
$To whom correspondence should be addressed: Laboratory of Western Blot Analysis-Immunoblots were performed as described
Pathology, National Cancer Institute, National Institutes of Health, previously (11, 12) with 4-chloro-1-naphthol as developing agent. Pri-
Bldg. 10, Rm.2A33, Bethesda, MD 20892.Tel.:301-496-8906;Fax: mary antibody was antipeptide 102 antibody diluted as described. Sec-
301-480-0853. ondary antibody was horseradish peroxidase-conjugated goat anti-rab-
The abbreviations used are: ATX, autotaxin; PCR, polymerasechain bit immunoglobulin (Pierce) diluted 1:2500.
reaction; 5‘RACE, rapid amplification of cDNA ends; Bistris, 2-[bis(2- Preparation of a Agtll cDNA Expression Library-Oligo(dT)-selected
hydroxyethyl~aminol-2-~hydroxymethyl)-propane-l,3-diol. poly(dA) mRNA was prepared from A2058 cells using standard tech-

30479

This is an Open Access article under the CC BY license.

30480 cDNA Clone for the Motility Factor Autotaxin
niques. The mRNA >lo00 base pairs were then purified from an agarose TABLEI
gel and used to prepare double-stranded cDNA using thecommercially Peptide sequences for autotaxin
available cDNA synthesis system from Promega. The cDNA inserts
Peptide Amino acid sequence
were synthesized using Not1 adapters andmodified with EcoRI linkers.
This allowed directional ligation into EcoRIINotI-digested Agtll Sfi-Not ATX- 18 WHVAXN
vector (Promega). ATX- 19 PXLDVYK
Antibody Screening of Agtll cDNA Expression Library-Approx- ATX-20 YPAFK
imately 5 x lo5 phage were screened by infecting LE 392 cells with the ATX-29 PEEVTRPNYL
recombinant Agtll followed by growth on LB agar plates (13). Phage ATX-34B RVWNYFQR
were transferred onto isopropyl-P-D-thiogalactoside-impregnated nitro- ATX-41 HLLYGRPAVLY
cellulose membranes by overnight incubation at 37 "C. The antibody ATX-47 YDVPFDAT
ATX-48 SNPPFENINLY
screening was performed using a modification of previously described ATX-59 TFPNLYTFLATGGLYW
immunoblottingtechniques (11, 12). In brief, themembranes were ATX-100 XGGQPLWITATK
blocked for 30 min inblocking buffer (1M glycine, 5% (w/v) nonfat dry ATX-l01/223A VNSMQTVFVGYGF'TFK
milk, 5%(v/v) fetal bovine serum, and1% (w/v) ovalbumin). The affinity ATX-102 DIEHLTSLDFFR
purified anti-ATX-102 antibody was incubated with the membranes in ATX-103 TEFLSNYLTNVD-
blocking buffer for 2 h at room temperature, using a concentration of DITLVPGTLGR
antibody whichwas double that which gavea strong response on West- ATX-104 VNVISGPIFDYDYDGLHDTEDK
ern blot analysis. Secondary antibody was horseradish peroxidase-con- ATX-204 MHTARVRD
jugated goat anti-rabbit immunoglobulin, and the blot was developed ATX-205 FSNNAKYD
colorimetrically with 4-chloro-1-naphthol. Positive phage were selected ATX-209 VMPNIEK
and purified repeatedly using the samedetection techniques until ho- ATX-210 TARGWECT
mogeneously pure. The positive reactivities of these clones were con- ATX-212 XDSPWT(N)ISGS
firmed by competition for antibody by specific versus unrelated pep- ATX-214 LRSCGTHXPYM
tides. cDNA inserts were subcloned into pBluescript (Stratagene) for ATX-215/34A TYLHTYES
further analysis. ATX-213/217A AIIANLTCKKPDQ
ATX-216 IVGQLMDG
5' Extension of the ATX cDNA Clone-A reverse transcriptase reac-
ATX-218/44 TSRSYPEILTPL
tion was performed using total or oligo(dT) purified RNA from A2058 or ATX-223B/24 QAEVSSVPD
N-tera 2D1 cells as template and an antisense primerfrom the 5' end ATX-224 RCFELQEAGPPDDK
of 4 C l l (GCTCAGATAAGGAGGAAAGAG).This was followed by one or ATX-229 SYTSCCHDFDEL
two PCR amplifications of the resultant cDNA using the commercially ATX-230 XFNHQWGGQQP
available kit from Perkin Elmer and following the manufacturer's di- ATX-239 AAECVPA
rections. These PCR reactions utilized nested antisense primers from ATX-244/53 QMSYGFLFPPYLSSSP
4 C l l (GAATCCGTAGGACATCTGCTT and TGTAGGCCAAACAGT-
TCTGAC) as well a s degenerate, nested sense primers deduced from
ATX peptides: ATX-101 (AAYTCIATGCARACIGTI'ITYGTIG and which contained the motif: (N)XT/S.
TTYGTIGGITAYGGICCIACITTYAA),ATX-103 (AAYTAYCTIACMY- The NH,terminus of the 125-kDa ATX band wassuccessfully
GTIGAYGAYAT and GAYGAYATIACICTIGTICCIGGIAC), or ATX-224 sequencedfrom Immobilonm to yield the sequence: DSP-
(TGYTTYGARYTICARGARGCIGGICCICC).The amplified DNA was WT(N)I. This appears tobe identical to ATX-212, now the pre-
then purified from a polyacrylamide gel using standardprocedures and
ligated into the pCRTMplasmid using the TA cloning kit (Invitrogen
sumed amino terminus of the secreted protein. Apparently,the
Corp.) according to manufacturer's directions. NH,-terminal aspartic acid is oxidized in the electrophoretic
The 5' RACE kit was utilizedt o extend the5' end ofATX cDNAusing procedures, since yields from the sequencing were <lo%.
total RNA from N-tera 2D1 as template and a previously obtained Characterization of Antipeptide Am-102-Antibodies were
sequence as primer (GCTGTCTTCAAACACAGC).The 5' end of the raised in rabbits to the peptide designated ATX-102 and affln-
A2058-synthesized protein was obtained by using a previously obtained ity-purified on a peptide affinity column. The resulting anti-
sequence as primer (CTGGTGGCTGTAATCCATAGC)in a reverse tran-
ATX-102 antibody recognized a 120-kDa protein on Western
scriptase reaction with totalA2058 RNA as template, followed by PCR
amplification utilizing the 5' end of the N-tera 2D1 sequence (see "Re- blot analysis of partially purified ATX (Fig. 1,Eane 2). When a
sults") a s sense primer(CGTGAAGGCAAAGAGAACACG)and a nested 1000-fold molar excess of peptide ATX-102 was preincubated
antisense primer (GCTGTCTTCAAACACAGC). with theantibody for 1 h prior to reacting with the membrane,
DNA Sequencing-DNA sequencing was performed using dideoxy detection of the protein band disappeared, indicatingantibody
methodology (14) and P5S1dATP (DuPont NEN). specificity (Fig. 1, lane 3). This antibody appeared to recognize
Assay for Fype I Phosphodiesterase Enzymatic Activity-Phospho- only denatured protein; it neither neutralized motility-stimu-
diesterase activity was measured according to Razzell (15) with minor
modifications. Samples were assayed in a 100-pl volume containing 50 lating activity nor immunoprecipitated native ATX.
mM Tris-HC1, pH 8.9, and 5 mM p-nitrophenyl thymidine-5'-monophos- Cloning of the ATX cDNA-Affinity-purified antipeptide
phate. After a 30-min incubationat 37 "C the reactions were terminated ATX-102 antibody was used toscreen an A2058 cDNA expres-
by addition of 900 1.11 of 0.1 N NaOH and the amountof product formed sion library. Eight purified cDNA clones were obtained that
was determined by reading the absorbance at 410 nm. reactedwith antibody in a peptide-specific manner. These
clones were each sequenced and one clone, designated 4Cl1,
RESULTS appeared to contain the3' terminus of ATX (Fig. 2 and Fig. 3).
Peptide Sequences of Purified ATX-Three separate homoge- The 4Cllclone contained 1084base pairs,including the polya-
neously pure protein preparations were digestedwith cyanogen denylated tail and the AATAAA polyadenylation signal motif.
bromide followed by trypsin (ATX-18 to ATX-591, trypsin alone The open reading frame region was 628 base pairs long and
(ATX-100 t o ATX-104), or with endo Lys-C (ATX-204 t o ATX- coded for 209 aminoacids.Within this coding region were
244). Purification of the fragments andsequence analysis were matches for 8 previously identified ATX peptides: ATX-20, ATX-
performed as described previously (3). A total of 30 peptides 34, ATX-102,ATX-104,ATX-204,ATX-215,ATX-218144, and
containing 315 amino acid residues have now been sequenced ATX-244. Northern blot analysis gave a weak band at -3.3
(Table I). The partial amino acid sequence of ATX did not ex- kilobase pairs, indicating that thisclone represented approxi-
hibit homology to known growth factorsor previously described mately one-thirdof the totalmRNA length. Data base analysis
motility factors. Several peptides were sequenced from two of of the 4Cllclone revealed a 45% amino acid identity anda 57%
the three purifications (shown by double numbers in thepep- nucleotide identity with a human nucleotide pyrophosphatase
tide name). In addition, 2 peptides (ATX 212 and ATX-2131 and kinase, PC-1, found on the surface of activated B cells and
217A) hadapparentasparagine-linked glycosylation sites plasma cells. This homology was utilized t o estimate the rela-
 cDNA Clone for the Motility Factor Autotaxin 30481
This was followedby one or two PCR amplificationsusing
281,300 + nested primers deduced from ATX-101, ATX-103,and ATX-224
(Fig. 2). In order to obtain the cDNA sequence encoding the
amino terminus of the protein, 5' RACE methodology was uti-
lized and succeeded in extending the5' end of the cDNA prod-
100,500 + uct from N-tera 2D12 but not from A2058 ATX. An oligonucleo-
tide with sequence matching the 5' terminus of this N-tera2D1
71,800 + cDNA clone was then utilized in a reverse transcriptasePCR
amplification to obtain the 5' end sequence of A2058 ATX
43,200 + cDNA. The cDNA and deduced protein sequence is shown in
Fig. 3. The total lengthof the cDNA is 3251 base pairs,roughly
equivalent to the length of the mRNA seen on Northern blot
28,500 analysis. I t includes an apparent initiating methionine sur-
rounded by a partial Kozak consensus sequence (16).
This deduced sequencehas been shown to matchall 30 of the
previously sequenced peptides (Table I, Fig. 3). For 25 of these
peptides, the match is exact. In the four peptides ATX-230,
1 2 3 ATX-19,ATX-48, and ATX-18, technical difficulties caused
FIG.1. Immunoblots of ATX with antipeptide ATX-102 anti- some misreadings of the sequence data. For peptide ATX-59,
body. Antipeptide antibodies were produced in rabbits by injecting
peptide ATX-102 conjugated to the carrier, bovine serum albumin. The the glutamine in the 10th aminoacid position appeared t o be
resultant antiserum was purified on an peptide affinity column. Par- correct and could represent a variation insplicing. In summary,
tially purifiedATX was then separatedby SDS-polyacrylamidegel elec- the peptide data provides strong evidence that we have se-
trophoresis run under nonreducing conditions and transferred electro-quenced the correct cDNA for purified ATX.
phoreticallyto an ImmobilonT" membrane. The affinity-purified
antipeptideantibody recognized abroad120-kDaproteinband on Protein Domainsand Homologies-Searches of protein data
immunoblot (lane 2). When the antibody was preincubated with 1000- bases (17)confirmed that thehomology between ATX and PC-1
fold molar excess of peptide for 1 h prior to reacting with the immuno- was present throughout the length of the extracellular portion
blot, the protein band was not detectable (lane 3 ). Prestained molecular of the molecules (18, 19). There is a 45% amino acid identity
weight markers are shownfor comparison in lane 1.
and a 64%similarity between the 2 protein sequences (Fig.4).
4C11 For the cDNA sequence, the identity is -57%.
103 224
P These proteins share several interesting properties anddo-
I I
W A ?. V V V mains (Fig. 5). Both have a number of potential N-linked gly-
S"UTR 3'-UTR cosylation sites: four for ATX (Asd4, Asn8") and
nine for PC-1. Both have adjacent somatomedin B domains
near the amino endof the extracellular domain. This somato-
medin B domain is a cysteine-rich region containing 3 pre-
sumed cystine cross-linkages. ATX has 33 Cys residues and
PC-1 has 37; 30 of these Cys residues are identical in place-
ment. Both proteins also contain theloop region of an EF-hand
(18, 20). In addition, both proteins have a transmembrane/
signal peptide region with a short intracellular peptide, com-
mon in ectoenzymes (21). However, the amino acid identity
between ATX and PC-1 in the intracellular and transmem-
brane regions is only 11%.
Finally, bothproteins havea region homologous to thebovine
intestinal phosphodiesterase enzymatic domain with conserva-
tion of the threonine thatis thought to act as the intermediate
phosphate binding site (22). PC-1 has been demonstrated to
have phosphodiesterase type I, nucleotidepyrophosphatase,
and threonine-specific kinase enzymatic activities (23, 24). In
I I I order to test whetherpurified ATX had type I phosphodiester-
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ase activity, samples were incubated with p-nitrophenyl thymi-
FIG.2. Cloning strategy for ATX cDNA. The cDNA clone, 4Cl1, dine-5'-monophosphate a t pH 8.9 for 30 min. ATX was found to
was selected from an A2058 expression library using the antipeptide hydrolyze the p-nitrophenyl thymidine-5'-monophosphate(15)
ATX-102 antibody a s a probe. This sequence was then utilized to syn- a t a rate of 10 pmol/ng/min, a reaction rate similar to that
thesize primersfor a reverse transcriptase reaction usingA2058 total or reported for PC-1 (25).
oligo(dT)-purified RNA as template. Nested primers from 4Cll and
previously sequenced ATX peptides (ATX-224,ATX-103, and ATX-101)
were utilizedfor PCR amplificationof the reverse transcriptase reaction DISCUSSION
product (as described under "Experimental Procedures"). 5' RACE was We have sequenced the cDNA and deduced the primary
utilized to obtain the far 5' end in N-tera 2D1cells and this sequence amino acid structure of ATX, a tumor cell motility-stimulating
was confirmed in A2058 cDNA. The arrows show the direction of DNA
sequencing. The scale indicates nucleotide positions in kilobases (hb). protein. Protein data base searches
of this sequence revealed a
UTR,untranslated region. 45% amino acid identitywiththeplasma cell membrane
marker protein,PC-1. ATX and PC-1 appear to share a number
tive positions of several of the largerATX peptides. of domains, including two somatomedin B domains, the loop
The 4 C l l clone was extended using reverse transcriptase/ region of anEF-hand,andtheenzymaticsite of type I
PCR methodology. The reverse transcriptasereaction was per-
formed using the 5' end of 4 C l l a s primer and total or oli- * Stracke, M. L., Arestad, A. A., Levine, M. D., Krutzsch, H. C. and
go(dT)-purified RNA from A2058 or N-tera 2D1 a s template. Liotta, L. A., Melanoma Res., in press.
30482 cDNA Clone for the Motility Factor Autotaxin

8
74 533
1649
33
149 558
1724
58
224 583
1799
83
299 608
1874
108
374 633
1949
133
449 658
2024
158
524 683
2099
183
599 708
2174
208
674 733
2249
233
749 758
2324
258
824 783
2399
283
899 808
2474
308
974 833
2549
333
1049 858
2624
358
1124 883
2699
383
1199 908
2774
408
1274 915
2849
433
1349 2924

458 2999
1424
3074
483
1499 3149

508 3224
1574

FIG.3. Nucleotide and deduced amino acid sequence of ATX The combined nucleotide sequence of four cDNA clones and their deduced
amino acid sequence is shown. The solid tine above the amino acid sequence indicates regions that match peptides previously sequenced by
enzymatic digestion and Edman degradation of purified autotaxin. Regions of peptide overlap are indicated by a heavier line.

phosphodiesteraselnucleotide pyrophosphatase. Like PC-1,
ATX hydrolyzes p-nitrophenyl thymidine-5'-monophosphate,a
type I phosphodiesterase substrate. This enzymatic function
suggests a newly identified function for ectolexo-enzymes in
cellular motility.
ATX has been previously purified from human melanoma cell
conditioned medium by testing fractions of sequential chro-
matographic separationsfor their capacity to stimulatecellular
motility (3). ATX was demonstrated to be a 125-kDa glycopro-
tein whose molecular mass reduced to 100-105 kDa after de-
glycosylation with N-glycosidase F.' The calculated molecular
mass of the cloned protein is 100 kDa (secreted form) or 105
kDa (full-length protein). Based on amino acid composition, the
estimated PI is 9.0 which is higher than the PI determinedby
two-dimensional gel electrophoretic analysis (7.7-8.0) of puri-
fied ATX. This could perhaps be explained by the presence of
sialic acid residues on the sugarmoieties. In addition, 25 out of
30 peptides, which had been sequenced from purified ATX by
Edman degradation,perfectly matched thededuced amino acid
sequence of the clone; the other 5 peptides had reasonable
matches. Taken together,these dataprovide good evidence that
we have sequenced the appropriate clone for ATX.
The homology with PC-1 was unexpected but continued
throughout the extracellular portion of the proteins (Fig. 5).
Both have adjacent somatomedin B domains near the amino
terminus of their extracellular portions. Somatomedin B, de-
rived from the amino terminus of vitronectin, forms the pre-
sumed binding site for type 1 plasminogen activator inhibitor
(26, 27). In extracellular matrix and in plasma, vitronectin is
thought to be the primary binding protein of activated plas-
minogen activator inhibitor (26). Although PC-1 has not been
shown to bind t o plasminogen activator inhibitor, this homol-
ogy does suggest a kinship with extracellular matrixproteins.
In addition, bothATX and PC-1 have regions homologous to the
active site of bovine type I phosphodiesterase (22) and both
hydrolyze the typeI phosphodiesterase substrate,p-nitrophen-
yl thymidine-5'-monophosphate.ATX and PC-1 also both con-
tain theloop region of an EF-hand,which is a calcium binding
domain that structurallyforms a helix-loop-helix (20). The loop
region is the actualcalcium binding site. Other proteins which
lack one or both helical regions are variablycapable of binding
calcium (20); however, this binding hasnot been demonstrated
for either PC-1 or ATX.
Despite the similarities, there are a number of important
differences between ATX and PC-1. First, the intracellular re-
gion ofATX is only 11amino acidslong and is different from the
26 intracellular amino acidsfound in PC-1. Likewise, the trans-
membrane domains are dissimilar. In addition, PC-1 exists in
the plasma membrane as a homodimer. Soluble monomeric
forms of PC-1 have been demonstrated in supernatants of plas-
macytoma cells, in transfected mouse L cells, and in normal
mouse serum (28). However, the presumedcleavage site of
PC-1, when the soluble form is generated, is between Arg16' and
Ala170,i.e. between the second somatomedin B domain and the
phosphodiesterase active site (28). In contrast,ATX appears to
be cleaved between Ser48 and Asp4', proximal to both somato-
medin B domains. Therefore,the soluble forms of the two mol-
ecules appear to be substantially different.
It remains unanswered how the domain structure of ATX
affects its capacity tostimulate motility. Other ectolexo-
enzymes have been shown to affect cellular motility or cellular
interactions with the extracellular matrix. For example, the
 Motility
Factor
Autotaxin
the for
Clone
cDNA 30483

hATX
JI
M A R R S S F Q S C Q I I S L F * P F A V O V S I C L G F * P A H R I K R A E O W E E C P P T V L ~ D ~ P W D9 0F
""""

""_
I I IIII I II I
hPCl
II
MDVGEEPLEKAARARTAKDPNTYKVLSLVLSVCVLTTIL
I I
........OCIFG ....LKP8CAKEVK.SCKGRCF...ERTFONCRCDMCVEU3NCCLDY
I 1 IIIIIII
"""_. 84
"

hATX D E L C L ~ ~ G ~ T ~ ~ ~ G E E ~ A C H C S ~ ~ G ~ ~ ~ C K G E S ~ D ~ E E I ~ P A G W R P P L I I F S M O F R A ~ Y ~1 9I 0I ( K G S K V
I I I I I IIII I I II II I I I I I1 I I I II II I IIIII II II IIIII I I I
hPCl

hATX
QE~:IEPEHIWTCNKFRCGERRLTRSLCACSDDCKDRODCCINYS~VCQOEK~WVEEPCE~INEPQCP~FETPPT~LFSLDCP
" YLHTWOOLLWIS184
Y
K L R S C G T H S P Y M R P W P T K T F P N L Y T L A T G L Y P E S H G I V G N ~ D ~ D A T F H L R G R E K ~ ~ P L W I T A T K ~ T F ~ S . . . . . . . . . .272
...
II Ill 1111l1l11111 I IIIIIIIIII I IIII I I I I I I I I I I I II I I I I I I I I
hPCl KLKKCGmTKNMRrmYPTKTFPNHYSIVTGLYPESHGIIDNlfmDPKMNA8FSLKSKEK~P~KGEPI~~~LKSGTF~S~IN GIFPDI
284

hATX .....WIPHERRILTILRWLTLPDHERPSVYAFYSEQPDFSGHKYGPFGPEESSYGSPPPPAltRPRRKVAPKRRQERWAPPRKR 372

I I Ill I II II Ill I I I II Ill I l l I
hPC 1 YKMYNGSVPFEERILAVLQQLPKDERPHPYTLYLEEPDSSGH~GWSSE ................................................ 336

hATX RQDKMTNPLREIDKIVGQLMDGLKQLKLRIFVGDHGMEDVTCDRTEFLSL~DITLVPOTLGRIR.SKF~.AKYDPKAIIANLTCKKPD 470

I I I I I I I I I I I I I1 I I IIIII I I I I I I I I l l I II I I
hPCl ....
VIKALQRVDGWVCMLMDGLKEHRCL~ILISDHGUEQOSCKKYIYLNKYLGWKNIKVIYGPMRLRPSDVPDRIYSFNYeOIARNLSCREPN432

hATX Q H F K P Y L K Q H L P K R L H Y A N N R R I E D I H L L V E R R W H V A R K P L D V Y K K P S G K C F F Q G D H G F D N ~ ~ ~ G Y G P T F K Y K T K V P P F ~570
I~~L~
IIIIIIII IIIIII I Ill I l l I I I IIII I I IIIIII II IIIII II IIIII
hPCl QHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSE RKYCGSGF .. ....H G S D N V P S N H Q A L F V G Y G P G F K H G I ~ F ~ I ~526
DL~

hATX L K P A P N N G T H G S L N H L L R T N F R P T M P E E V T R P N Y P C I M Y D . E L N K R L H T K G S T E E R H L L Y G R P A V L Y R T R . Y D I L Y H T668
I IIIIIIIIIIIIIII I I II I Ill I I I I IIII II I
hPCl LTPAPNNGTHGSLNHLLKNPTPKHPKEV.HPLVQCPFTVDRNPRD~GCSCNPSILPIEDFQTQ~L~~EKIIKH~LPYGRPR~K~1CLLSQH 625

hATX D F E S G Y S E I F L M L L ~ S Y ~ S K Q A E V S S V P D H L T S ~ P D ~ V S P S F S Q N C L A Y K N D K Q M S Y G F L F P P Y L S S S P ~ . D ~767
L~P~P~KR~
I IIII II IIIIIII I I I I I I Ill I I I I I II I I I I I I Ill1 I I I
hPCl QFMSGYSQDILMPL~SYTVDRNDSFS..TEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVmGFLSPPQLNKNSSGIYSEALLT 723

hATX FQRVLVKKYASERNGVNVISGPIFDYDYDGLHDTEDKIKQ.. .WEGSSIPVPTHYYSIITSCLDFTQPADKCDGPLSVSSFILPHRPDNEE~SSEDE875

I I Ill IIIIIII Ill II Ill1 I I I Ill Ill I I I I IIIIII II Ill
hPCl FHDTLLRKY~ERNGVNWSGPVFDFDYDCRCDSLENLRQKRRVI~QEILIPTHFFIVLTSCKDTSQTPLHCPl.LDTLAFILPHRTDNSE8CVHCKHD
822

hATX SKWVEELMKMHTARVRDIEHLTSLDFFRKTSRSYPEILTLKTYLHTYESEI915
I IIIII I I I I II I I I I I I I I I I I
h P C l SSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED 873
FIG.4. Comparison of amino acid sequences of ATX and PC-1. The amino acid sequences of ATX and PC-1 are compared. Amino acid
identity is indicated by a vertical line between the sequences. The locationof the putative transmembranehignal sequence is shown by solid lines.
The two somatomedin B domains are identified by dashed lines. The putative phosphodiesterase active site is indicated by emboldened lines. The
loop region of a single EF-hand loop region is identified with double lines. The presumed cleavage site for each protein is indicated with arrows.

Ex tra c e l l ul a r Thus, specific intracellular enzymes have been shown to be

Cleavage Slter
chemotactic when placed in an extracellular environment.
If ATX isanextracellular kinase/phosphodiesterase like

\
PC-1, its capacity t o modulate protein phosphorylation could

\
affect multiple cell surface proteins as well as components of
the extracellular matrix. Such a capacity would suggest that
/
region Phosphodleslerase
Active Site
"Loop" ATX could play a regulatory role in the interaction of the mi-
intracellular
grating cell with its microenvironment as well as a direct role
Somatomedin E in the receptor-mediated stimulation of motility.
domains
Tranmembrarm
dmah Acknowledgments-We thank Dr. Elliott Schiffmann for his constant
moral and intellectual support. We also thank Dr. Richard E. Manrow
FIG.5. Domain structure of ATX and PC-1.Putative domains are for his help and technical suggestions in the cloning of the autotaxin
indicated for the two homologous proteins, ATX and PC-1. cDNA.

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