thewnt/planarcellpolarityprotein-tyrosinekinase-7(ptk7 ... · defects in the non-canonical wnt/pcp...

The Wnt/Planar Cell Polarity Protein-tyrosine Kinase-7 (PTK7)Is a Highly Efficient Proteolytic Target of Membrane Type-1Matrix MetalloproteinaseIMPLICATIONS IN CANCER AND EMBRYOGENESIS*

Received for publication, July 15, 2010, and in revised form, September 7, 2010 Published, JBC Papers in Press, September 13, 2010, DOI 10.1074/jbc.M110.165159

Vladislav S. Golubkov, Alexei V. Chekanov, Piotr Cieplak, Alexander E. Aleshin, Andrei V. Chernov, Wenhong Zhu,Ilian A. Radichev, Danhua Zhang, P. Duc Dong, and Alex Y. Strongin1

From the Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037

PTK7 is an essential component of theWnt/planar cell polar-ity (PCP) pathway. We provide evidence that the Wnt/PCPpathway converges with pericellular proteolysis in both normaldevelopment and cancer. Here, we demonstrate thatmembranetype-1 matrix metalloproteinase (MT1-MMP), a key proinva-sive proteinase, functions as a principal sheddase of PTK7.MT1-MMP directly cleaves the exposed PKP6212LI sequenceof the seventh Ig-like domainof the full-lengthmembranePTK7and generates, as a result, an N-terminal, soluble PTK7 frag-ment (sPTK7). The enforced expression of membrane PTK7 incancer cells leads to the actin cytoskeleton reorganization andthe inhibition of cell invasion. MT1-MMP silencing and theanalysis of the uncleavable L622D PTK7 mutant confirm thesignificance ofMT1-MMPproteolysis of PTK7 in cell functions.Our data also demonstrate that a fine balance between the met-alloproteinase activity and PTK7 levels is required for normaldevelopment of zebrafish (Danio rerio). Aberration of this bal-ance by the proteinase inhibition or PTK7 silencing results inthe PCP-dependent convergent extension defects in thezebrafish. Overall, our data suggest that the MT1-MMP-PTK7axis plays an important role in both cancer cell invasion andnormal embryogenesis in vertebrates. Further insight into thesenovel mechanisms may promote understanding of directionalcell motility and lead to the identification of therapeutics totreat PCP-related developmental disorders and malignancy.

Secreted Wnt glycoproteins regulate �-catenin-dependent(canonical) and �-catenin-independent (non-canonical) sig-naling pathways (1–7).One intriguing andwell conserved func-tion of the non-canonical pathway is to control PCP2 and direc-tional cell motility (8). PCP governs the orientation of cells in amonolayer of a tissue plane (front-back orientation) in such a

way that all cells within the monolayer are aligned in the samedirection. As a result, PCP is important for the directed collec-tive cell movements and orchestrates the synchronized cellarrangements within the tissue plane in the course of a plethoraof biological processes (2, 4, 6–11).The first PCP signaling events occur at a gastrulation stage of

embryogenesis to regulate the polarized cell movement andaccomplish convergent extension (CE) for the anterior-poste-rior body axis elongation, neural tube closure, and craniofacialmorphogenesis (8, 9, 11, 12). CE failure results in the multiplesevere developmental defects, including a shortened body axis(dwarfism), defective neural system, and craniofacial abnor-malities. Defects in the non-canonical Wnt/PCP pathway arelinked to a broad range of diseases, including cancer (3, 5).Wnt5a, Wnt5b, and Wnt11, which work through the non-ca-nonical pathway, are often up-regulated in cancer and promotecancer cell motility and invasion (6, 13). Evidently, an in depthmechanistic understanding of the PCPmechanismand its aber-rant regulation in disease is required to control tumor progres-sion and metastasis in a clinically advantageous manner (6).Human PTK7 pseudokinase (also known as colon carcinoma

kinase-4, CCK-4) is required for PCP and CE (14–16). Thefull-length membrane PTK7 receptor consists of seven extra-cellular Ig domains, a transmembrane region, and a catalyticallyinert cytoplasmic tyrosine kinase (PTK) domain (17–20). PTK7is evolutionary conserved, and its orthologs include mousePTK7, chicken KLG, Drosophila Dtrk/Off-track (OTK), andHydra Lemon (21). PTK7 mutant mice that expressed a 1–114PTK7 truncation died perinatally with severe defects in neuraltube closure, a CE process (16). Overexpression of the mutantPTK7 lacking its cytoplasmic domain resulted in similar abnor-malities. An N-terminal, soluble PTK7 fragment (sPTK7)inhibited angiogenesis in vitro and in vivo in a dominant nega-tive fashion by competing with the full-length PTK7 (22). Theexpression of PTK7 is frequently deregulated in cancers (4,23–25).Directional cell locomotion is highly dependent on both well

orchestrated actin cytoskeleton dynamics and efficient pericel-lular proteolysis (26, 27). Proinvasive, promigratory MT1-MMP (MMP-14), a prototypic member of theMMP family, is amajormediator of pericellular proteolytic events in cancer cells(28). MT1-MMP cleaves ECM proteins, initiates activation ofsoluble MMPs, and controls the functionality of cell adhesionand signaling receptors. MT1-MMP is a prototypic member of

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA83017 and CA77470 (to A. Y. S.).

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) GU211905.

1 To whom correspondence should be addressed. Tel.: 858-795-5271; Fax:858-795-5225; E-mail: [email protected].

2 The abbreviations used are: PCP, planar cell polarity; CE, convergent exten-sion; MLC, myosin light chain; pMLC, phosphorylated MLC; MMP, matrixmetalloproteinase; MT1-MMP, membrane type-1 matrix metalloprotein-ase; PTK, protein-tyrosine kinase; sPTK7, N-terminal, soluble PTK7 frag-ment; TIMP, tissue inhibitor of metalloproteinases; MDCK, Madin-Darbycanine kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 46, pp. 35740 –35749, November 12, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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a membrane-anchored MMP subfamily and is distinguishedfrom soluble MMPs by a C-terminal transmembrane domainand a cytoplasmic tail (29–31). MT1-MMP is synthesized as alatent zymogen that requires proteolytic processing of theN-terminal inhibitory prodomain (32, 33). Once activated,MT1-MMP can be inhibited by its physiological inhibitors, tis-sue inhibitors of metalloproteinases-2, -3, and -4 (TIMP-2, -3,and -4). In contrast, TIMP-1 is a poor inhibitor of MT1-MMP(34, 35).MT1-MMP, as opposed to the solubleMMPs, is ideally posi-

tioned to regulate pericellular proteolysis and the functionalityof cell receptors (36). In migrating cells, MT1-MMP accumu-lates predominantly at the leading and trailing edges and, as aresult, contributes most efficiently to cell locomotion (37–39).Knock-out ofMT1-MMPhas themost significant phenotype

among MMP gene knock-out mice; MT1-MMP knock-outmice are dwarfs and die at adulthood (40, 41). Likewise, a loss ofthe structurally similar primordial At2-MMP induces dwarfismin Arabidopsis plants (42).

Recent studies link MT1-MMP to the non-canonical Wnt/PCP pathway in embryogenesis and cancer (27, 43). Both tran-scriptional silencing and enforced overexpression of MT1-MMP negatively impacted CE and craniofacial morphogenesisin zebrafish, suggesting that a stringent control of MT1-MMPactivity is essential innormaldevelopment (27, 44, 45).Themolec-ular mechanisms involved in the MT1-MMP-dependent regula-tion of the non-canonical Wnt/PCP signaling pathway, however,remain elusive. Intriguingly, co-expression data of 19,777 humanand 21,036 mouse genes from the COEXPRESdb database indi-cate thatMT1-MMP and PTK7 are closely co-expressed.Here, we provide evidence that the full-length membrane

PTK7 affects actin cytoskeleton and inhibits cancer cell inva-sion. Our results demonstrate that MT1-MMP directly cleavesthe full-length membrane PTK7, that this cleavage generatesthe sPTK7 species, and, most importantly, that this proteolyticevent is ubiquitous inmultiple cell systems. Taken together, ourexperimental data suggest that the pericellular proteolysis andPCP mechanisms converge in the regulation of directional cellmigration and that they work in concert in processes as diverseas embryogenesis and malignancy.

MATERIALS AND METHODS

Antibodies, Reagents, and Cells—A rabbit polyclonal anti-body against the N-terminal portion of PTK7 was a kind gift ofDr. Xiaowei Lu (University of Virginia, Charlottesville, VA). Agoat polyclonal antibody (catalog no. AF4499) against the

N-terminal 31–199 portion of PTK7 was from R&D. A murinemonoclonal 3G4 antibody (catalog no. MAB1767) against thecatalytic domain of MT1-MMP, a rabbit AB8104 antibody tothe hinge domain of MT1-MMP, and the GM6001 hydroxam-ate inhibitor were from Chemicon. Amurine monoclonal anti-body to the V5 tag was from Invitrogen. A murine monoclonalFLAGM2 antibody, the FLAGM2 antibody-agarose beads, anda polyclonal rabbit TGN46 antibody (catalog no. T7576) werefrom Sigma. The phosphomyosin light chain 2 (Ser19) rabbitpolyclonal antibody (catalog no. 3671) was from Cell Signaling.Rhotekin-RBD agarose beads and a RhoAmonoclonal antibody(catalog no. ARH01) were from Cytoskeleton. EZ-Link sulfos-uccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate wasfrom Pierce. Human fibrosarcoma HT1080, breast carcinomaMCF7, mammary epithelial MCF10A and 184B5 cells, andMadin-Darby canine kidney (MDCK) cells were from ATCC(Manassas, VA). A highly metastatic M4A4 clone of breast car-cinoma MDA-MB-435 cells was a gift of Dr. Virginia Urquidi(University of California, San Diego, CA). MCF10A and 184B5cells were grown in mammary epithelial growth medium(Lonza). Other cell lines were grown in DMEM supplementedwith 10% FBS. The recombinant catalytic domain of MT1-MMP was characterized earlier (46). The CD44 (catalog no.3570) and E-cadherin (catalog no. 610181) monoclonal anti-bodies were obtained from Cell Signaling and BD Biosciences,respectively.Cloning and Mutagenesis—MCF7, HT1080, 184B5, MDA-

MB-435, and MDCK cells transfected with the full-lengthMT1-MMP (MCF7-MT1, HT1080-MT1, 184B5-MT1, MDA-MB-435-MT1, and MDCK-MT1 cells, respectively) wereestablished and characterized earlier (47, 48). HT1080 cellswith the over 90% transcriptional silencing of MT1-MMP andthe required scrambled controls were obtained and extensivelycharacterized earlier (47, 49, 50). The full-length wild-typePTK7 cDNA (OriGene) was amplified by the PCR using theselective PTK7 primers. The construct was subcloned into thepcDNA3.1D/V5-His-TOPOdirectional TOPOexpression vec-tor (Invitrogen). Where indicated, the PTK7 construct wasC-terminally tagged with the V5-His and FLAG tags. The full-length PTK7-FLAG template was used to generate the L622D,M641R, andM701Dmutants and the sPTK7 1–700 constructs.The primers we used in our experiments are shown in Table 1.The PTK7 constructs were used to stably transfect HT1080,MCF7, MDCK, MDA-MB-435, and MDA-MB-435-MT1 cellsusing Lipofectamine 2000 (Invitrogen). The full-length and the

TABLE 1Oligonucleotide primers used in our study

Construct Forward primer Reverse primer

PTK7 5�-CACCATGGGAGCTGCGCGGGGATC-3� 5�-TCACGGCTTGCTGTCCAC-3�PTK7-V5-His 5�-CACCATGGGAGCTGCGCGGGGATC-3� 5�-CGGCTTGCTGTCCAC-3�PTK7-FLAG 5�-CACCATGGGAGCTGCGCGGGGATC-3� 5�-TCACTTGTCATCGTCGTCCTTGTAGTCCGGCTTGCTGTCCACGGTGC-3�sPTK7 5�-CACCATGGGAGCTGCGCGGGGATC-3� 5�-TCACTTGTAGGGGGGAGGGCTGCCAG-3�sPTK7-V5-His 5�-CACCATGGGAGCTGCGCGGGGATC-3� 5�-CTTGTAGGGGGGAGGGCTGCCAG-3�sPTK7-FLAG 5�-CACCATGGGAGCTGCGCGGGGATC-3� 5�-TCACTTGTCATCGTCGTCCTTGTAGTCCTTGTAGGGGGGAGGGCTGCCAG-3�L622D 5�-GGGGACCCCAAGCCGGATATTCAGTGGAAAGGC-3� 5�-GCCTTTCCACTGAATATCCGGCTTGGGGTCCCC-3�M641R 5�-CCAAGCTGGGACCCAGGCGGCAC-3� ATCTTCC 5�-GGAAGATGTGCCGCCTGGGTCCCAGCTTGGM701D 5�-CCTCCCCCCTACAAGGATATCCAGACCATTGGG-3� 5�-CCCAATGGTCTGGATATCCTTGTAGGGGGGAGG-3�Zebrafish PKT7 5�-GCGACCACAACATCACACTC-3� 5�-TCCATCACTCAGCTCAGCAC-3�Zebrafish PKT7 5�-GGATCAACAGTGCTGAGCTG-3� 5�-CAGACTCTTGACCAGCACCA-3�

Proteolysis and Non-canonical Wnt Signaling

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soluble PTK7 constructs were also transfected into MDCK-MT1 cells to generate the doubly transfected MDCK-MT1-PTK7 andMDCK-MT1-sPTK7 cells, respectively. HT1080 sta-bly transfected with MT6-MMP were used as an additionalcontrol in our study (51).Cell Surface Biotinylation, Two-dimensional PAGE, and Pro-

tein Identification by LC/MS/MS—Cell surface proteins werebiotinylated by incubating cells for 1 h on ice in PBS containing0.1 mg/ml EZ-Link sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate. Cells were lysed in 20 mM Tris-HCl, 150mMNaCl, 1% deoxycholate, 1% IGEPAL, pH 7.4, supplementedwith a protease inhibitor mixture set III (Sigma), 1 mM phenyl-methylsulfonyl fluoride, and 10 mM EDTA. Biotinylated pro-teins were precipitated from cell lysates with streptavidin-aga-rose beads (Sigma). Biotinylated proteins were eluted from thebeads using 50mMDTT.The sampleswere then alkylated usinga ReadyPrep reduction-alkylation kit (Bio-Rad) and separatedby two-dimensional PAGE using a PROTEAN II xi Cell (Bio-Rad). The gels were stainedwith SimplyBlue SafeStain (Invitro-gen). The gel images were analyzed with ImageJ software(National Institutes of Health). The individual stained spotswere excised from the gel and subjected to in-gel trypsin diges-tion with Trypsin Gold, mass spectrometry grade (Promega).The digest samples were analyzed by LC/MS/MS using an LTQXL linear ion trap mass spectrometer (Thermo Scientific).MS/MS spectra were searched against the Swiss-Prot data baseusing SEQUEST Sorcerer software. The peptides with a proba-bility score of �0.95 and a cross-correlation (Xcorr) value of�2.0 were further analyzed and annotated.MT1-MMP Proteolysis of PTK7 in Vitro—The biotinylated

plasma membrane proteins from MCF7 cells (1 � 106) werecaptured on streptavidin-agarose beads (20 �l of a 50% slurry)and co-incubated for the indicated time at 37 °C with therecombinant catalytic domain of MT1-MMP (20 nM) in 100 �lof 50 mM HEPES, pH 6.8, containing 10 mM CaCl2, 0.5 mM

MgCl2, and 50 �M ZnCl2. The digests were analyzed by West-ern blotting with the PTK7, CD44, and E-cadherin antibodies.Modeling of the PTK7 Structure—The three-dimensional

structure of PTK7 was modeled by threading its sequence onthe known structures of the homologues using the programMODWEB (52, 53). The first six Ig domains of PTK7 (residues28–588) were built using Protein Data Bank entry 3B43 (titin)as a template. The seventh Ig domain of PTK7 (residues 594–684) was built from the fragment of Protein Data Bank entry2DM7 (Kiaa1556, residues 22–105, sequence identity 38%).The transmembrane region of PTK7 (residues 703–778) wasbuilt using Protein Data Bank entry 1SYS (Hla), and the PTK7kinase domain (residues 789–1072) was built using a fragmentof Protein Data Bank entry 2BDF (Src, residues 258–525,sequence identity 38%). The modeled fragments of PTK7 weremerged together and visualized by using PyMOL software(DeLano Scientific).Immunofluorescence—Cells grown on a glass coverslip were

fixedwith 4%paraformaldehyde for 10min, permeabilizedwith0.1% Triton X-100 for 5 min, and then blocked in 1% casein for1 h. Cells were stained with the primary antibodies (dilution1:1,000) for 16 h at 4 °C, followed by stainingwith the secondaryantibodies conjugated with AlexaFluor 488 or AlexaFluor 594

(Molecular Probes; dilution 1:500) for 2 h at ambient tempera-ture. The coverslips were mounted in the Vectashield mount-ing medium with DAPI (Vector Laboratories). Images wereacquired on an Olympus BX51 fluorescence microscopeequipped with aMagnaFire digital camera andMagnaFire 2.1Csoftware (Olympus).Rho Activation Assay—The Rho activation assay kit (Cy-

toskeleton)was used to estimate the cellularGTP-boundRhoA.Briefly, the active, GTP-bound RhoAwas precipitated from thecell lysate using rhotekin-RBD beads. The precipitated RhoAwas analyzed by Western blotting with a RhoA monoclonalantibody.Collagen Gel Contraction Assay—Cells (1 � 105) were mixed

with 0.2 ml of type I collagen in DMEM (0.3 mg/ml) on ice andplaced inwells of a 24-well low adhesion cell culture plate. Aftergel polymerization at 37 °C for 30min, the growthmedium (0.5ml) was added to the wells. Following a 24-h incubation, the gelimages were taken using a digital camera.Invasion Assay—The invasion assay was performed in wells

of a 24-well Transwell plate with an 8-�m pore size membrane(48). The membranes of Transwell inserts were coated withtype I collagen (2.5 �g/well; BD Bioscience). Cells (1 � 105/well) were placed in serum-free DMEM (0.1 ml) into the upperchamber. The 10% FBS-containingDMEM (used as a chemoat-tractant, 0.6 ml) was placed in the lower chamber. Serum-freeDMEM (0.6 ml) was used as a control. Cells were allowed toinvade for 6 h. The cells were then stained for 10 min with 0.2%crystal violet in 20% methanol (0.3 ml). The cells on the uppermembrane surface were removed with a cotton swab. The dyefrom the cells thatmigrated onto themembrane’s lower surfacewas extracted with 1% SDS (0.25ml). The resultingA570 nm wasmeasured using a SpectraFluor Plus plate reader (Tecan). Theassays were run in triplicate in three independent experiments.Immunohistochemistry—Breast cancer tissue arrays BR962

were from US Biomax. After deparaffinization and antigenretrieval, the arrays were stained using the goat PTK7 poly-clonal antibody and theMT1-MMP 3G4monoclonal antibody,followed by the staining with the secondary antibodies conju-gated with AlexaFluor 488 or AlexaFluor 594.Zebrafish Maintenance, Treatment with Inhibitors andMor-

pholino Injection—The zebrafish (AB strain) were maintainedunder the standard laboratory conditions at 28.5 °C. One-cellstage embryos were collected from natural matings. GM6001(100 �M) was added to the one-cell stage embryos and replacedtwice daily. MT1-MMP (MMP14a and MMP14b) and PTK7morpholinos 5�-GACGGTACTCAAGTCGGGACACAAA-3�and 5�-GAACCCGCTCCAGATCATTTTTCGC-3� (MT1-MMP) and 5�-GCTTGCTCTTGCTCTCTCCCGGCAT-3�(PTK7) were from Gene Tools. The morpholinos were micro-injected at a one-cell stage (2–10 ng/embryo). To examine pro-tein expression, the embryoswere lysed in 20mMTris-HCl, 150mMNaCl, 1% deoxycholate, 1% IGEPAL, pH 7.4, supplementedwith a protease inhibitor mixture set III (Sigma), 1 mM phenyl-methylsulfonyl fluoride, and 10 mM EDTA. The lysates wereanalyzed by Western blotting with the goat PTK7 antibody(R&D Systems).Whole Mount in Situ Hybridization—The in situ hybridiza-

tion of zebrafish embryos using the antisense RNAprobemyoD




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was performed following the protocol described previously(54).The Zebrafish PTK7 cDNA Sequence and RT-PCR—Five

48-h-old embryos were homogenized in 1 ml of TRIzol(Invitrogen) by passing through a 20-gauge needle. Total RNAsamples were extracted from the lysates and then purified usingthe RNA miniprep columns (Zymo Research). First-strandcDNA was synthesized using the purified RNA samples (1 �g),SuperScript II reverse transcriptase (Invitrogen), and a randomprimer (100 ng). The forward and reverse primers for thePCR amplification of the zebrafish Ptk7 cDNA fragments(5�-GCGACCACAACATCACACTC-3� and 5�-TCCATC-ACTCAGCTCAGCAC-3�, respectively, and 5�-GGATCA-ACAGTGCTGAGCTG-3� and 5�-CAGACTCTTGACCAG-CACCA-3�, respectively) were designed using Primer 3 software(55). These primer sets were also used for the RT-PCR. Theamplification reactions (25 �l) included the cDNA (50 ng)and the respective primers (0.6 �M). PCRs (30 cycles) wereperformed using denaturation at 95 °C for 30 s, annealing at58 °C for 30 s, and elongation at 72 °C for 1 min. The prod-ucts were separated by 2% agarose gel-electrophoresis. Spe-cific PCR products were purified from the gels using a gelextraction kit (Qiagen). Both the sense and antisense cDNAstrands were sequenced to obtain the nucleotide sequence ofPTK7. As a result, the 1782-bp cDNA sequence of zebrafishPTK7 was assembled and deposited in GenBankTM (acces-sion number GU211905).

RESULTS

PTK7 Is a Proteolytic Target of MT1-MMP—To search fornovel cell surface cleavage targets of MT1-MMP, we comparedtwo-dimensional gel profiles of biotin-labeled plasma mem-brane proteins from normal mammary 184B5 epithelial cellswith those from 184B5 cells stably transfected withMT1-MMP(184B5-MT1 cells) (Fig. 1). The identity of the protein spotswasdetermined using LC/MS/MS. We readily detected reducedlevels of PTK7 in 184B5-MT1 cells. We then determined PTK7levels in several normal and cancer cell lines of a diversifiedtissue origin (namely 184B5, MCF10A, MCF7, MDA-MB-435,HT1080, and MDCK) (Fig. 2A).Normal 184B5 and MCF10A mammary cells as well as non-

invasive breast carcinoma MCF7 cells exhibited high levels offull-length membrane PTK7. In contrast, PTK7 levels were lowin highly metastatic, invasive breast carcinoma MDA-MB-435cells and in 184B5 and MCF7 cells in which MT1-MMP hadbeen overexpressed (184B5-MT1 and MCF7-MT1 cells, re-spectively). Treatment of cells with GM6001 (a wide rangehydroxamateMMP inhibitor) or tissue inhibitor ofmetallopro-teinases-2 (TIMP-2; a potent MT1-MMP inhibitor) increasedlevels of full-length PTK7 in MCF7-MT1 cells to thoseobserved in MT1-MMP-deficient MCF7 cells. TIMP-1 (aninefficient MT1-MMP inhibitor) had no effect (Fig. 2B). MT1-MMP activity was correlated with the presence of sPTK7 in themedium. Expression of the lipid raft-associated MT6-MMP incells did not promote a similar effect (Fig. 2C). sPTK7 was alsodetected in plasma membrane samples, suggesting that sPTK7and full-length PTK7 interact (Fig. 2E).

In agreement with our two-dimensional gel profiling data,PTK7 was highly sensitive to MT1-MMP proteolysis in vitro,especially when compared with well known targets of MT1-MMP, such as CD44 and E-cadherin (56, 57) (Fig. 2D). Takentogether, our results indicate that PTK7 is a major target ofMT1-MMP.MT1-MMP Directly Cleaves the PKP6212LI Site of PTK7—

We next determined the identity of the cleavage site and thecleavage events that result in sPTK7. To predict PTK7 cleavagesite(s) that could lead to sPTK7 generation, we analyzed theregion encompassing PTK7 amino acids 600–710 for potentialMMP cleavage sites using softwarewe developed (58) (Table 2).We identified three potential MT1-MMP cleavage sites(PKP6212LI, PRM6412HI, and PYK7002MI) in this region.Corresponding L622D, M641R, and M701D PTK7 pointmutants with inactivated cleavage sites were generated, andthe constructs were expressed in fibrosarcomaHT1080 cells,which express significant levels of endogenous MT1-MMP(59).To support our results, mutants were also transfected into

MDA-MB-435 cells and MT1-MMP-expressing MDA-MB-435-MT1 cells. Our analysis determined that the L622Dmutant was fully resistant to MT1-MMP proteolysis in bothHT1080 and MDA-MB-435-MT1 cells. Structural modelingsuggests that the PKP6212LI cleavage site is localized in theexposed region of the seventh Ig-like domain of PTK7, and, as aresult, it is probably accessible to proteolysis (Fig. 3).MT1-MMP and PTK7 Co-localize in the Cells—The

observed proteolysis of PTK7 by MT1-MMP suggests that theproteinase and the kinase are proximal to each other in cells. As

FIGURE 1. MT1-MMP cleaves PTK7. Biotin-labeled plasma membrane pro-teins from normal mammary epithelial 184B5 cells transfected with MT1-MMP (184B5-MT1 cells) and untransfected control cells were separated bytwo-dimensional gel electrophoresis (bottom panels). The densitometry pro-file of the selected region (square) is shown in the top panels. The identity ofPTK7 (circled) was confirmed by LC/MS/MS.




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predicted, MT1-MMP and PTK7 co-localized at cell-cell junc-tions in all cell types analyzed, including 184B5 cells, whichco-express endogenous MT1-MMP and PTK7. Enforcedexpression of MT1-MMP in MDCK epithelial cells (MDCK-MT1 cells) resulted in reduced levels of full-length membranePTK7 both at cell-cell junctions and at the leading edge ofmigrating cells (Fig. 4,A–C). The C-terminal fragment of PTK7(anti-V5 staining) but not full-length PTK7 (anti-PTK7 stain-ing), however, accumulates at the leading edge in migratingMDCK-MT1 cells. These observations indicate the enhancedproteolysis of membrane PTK7 by MT1-MMP which, nor-mally, redistributes to the leading edge, and, as a result, MT1-MMP proteolysis contributes to the polarized localization ofmembrane PTK7 in migrating cells. Our findings support therole of PTK7 in cell migration and correlate with earlier obser-vations by others who showed that PTK7 contributes to neural

crest migration in Xenopus byrecruiting Dishevelled (14). Inagreement, Van Gogh-like 2,another important regulator of thenon-canonical Wnt pathway, alsoco-localizes with MT1-MMP andredistributes toward the leadingedge of the polarized human cancercells (27).Previous reports indicate that

PTK7 levels are inversely correlatedwith melanoma tumorigenicity(25). To further investigate the rela-tionship between PTK7 and cancer,we analyzed PTK7 in breast cancerbiopsies by immunostaining andobserved reduced membrane PTK7immunoreactivity in tumor lesionscompared with normal mammarytissue. In the analyzed tumors,MT1-MMP and PTK7 were co-localized at cell-cell junctions(Fig. 4D).MT1-MMP/PTK7 Axis Regulates

the Actomyosin Contractility andCell Invasion—Although previousreports indicate that PTK7 regu-lates PCP, the precise role of PTK7in the non-canonical Wnt/PCPsignaling is not known (14–17, 19).To determine such a role, we ana-lyzed actin cytoskeleton organiza-tion, RhoA GTPase activation, and

myosin light chain (MLC) phosphorylation (pMLC) in HT1080cells transfected withMT1-MMP siRNA and PTK7 expressionconstructs. Expression of the full-length PTK7 and the uncleav-able L622D PTK7 mutant altered the actin cytoskeleton, par-ticularly in membrane ruffles, and reduced levels of pMLCcompared with those seen in the parental HT1080 cells (Fig. 5,A and B). Similarly, silencing of MT1-MMP using siRNAdecreased pMLC levels. Conversely, expression of sPTK7 alonedramatically stimulated RhoA activity in cells (Fig. 5C).MLCphosphorylation is critical for actomyosin contractility,

which, in turn, correlates with the ability of cells to contractthree-dimensional collagen gels (60–62). In agreement, pMLClevels were positively correlated with the ability of cells to con-tract three-dimensional collagen gels in our cell system (Fig.5D). Because MT1-MMP expression and alterations in actincytoskeleton dynamics are known to be associated with cellinvasion (63, 64), we next analyzed the invasive capacity ofHT1080 cells that had been transfected with MT1-MMPsiRNA and PTK7 constructs. Cells transfected with full-lengthPTK7 and, particularly, with the uncleavable L622D constructwere significantly less invasive compared with HT1080 controlcells, whereas overexpression of sPTK7 did not promote anysignificant effect. Conversely, PTK7 proteolysis by MT1-MMPreversed this inhibitory effect (Fig. 5E).We concluded that full-length PTK7 strongly represses cell invasion and that PTK7

FIGURE 2. MT1-MMP directly cleaves the full-length membrane PTK7 and generates sPTK7. A, biotin-labeled plasma membrane proteins from normal mammary 184B5 and MCF10A epithelial cells, non-invasivebreast carcinoma MCF-7, metastatic breast carcinoma MDA-MB-435, and MT1-MMP-transfected cells (184B5-MT1 and MDA-MB-435-MT1) were analyzed by immunoblotting with the PTK7 antibody. B, GM6001 andTIMP-2 (but not TIMP-1) inhibit MT1-MMP proteolysis of membrane PTK7. Biotin-labeled plasma membraneproteins from MCF7 and MCF7-MT1 cells were analyzed by immunoblotting with the PTK7 antibody. C, sPTK7is released by MT1-MMP-transfected cells (HT1080-MT1) but not by MT6-MMP-transfected HT1080-MT6 cells.D, MT1-MMP cleaves PTK7 more efficiently than it does E-cadherin and CD44. Biotin-labeled plasma membraneproteins from MCF7 cells were co-incubated with the recombinant catalytic domain of MT1-MMP. Sampleswere analyzed by immunoblotting with PTK7, CD44, and E-cadherin antibodies. The right lanes show full-length PTK7 and soluble, N-terminal sPTK7 fragment controls (from MCF7-PTK7 and MCF7-sPTK7 cells, respec-tively). E, the N-terminal PTK7 fragment is present in both MDCK cells that co-express full-length PTK7 andMT1-MMP constructs (MDCK-MT1-PTK7 cells) and MDCK cells that co-express MT1-MMP with the soluble PTK7construct (MDCK-MT1-sPTK7 cells). Where indicated, GM6001 was added to the cells.

TABLE 2Potential MT1-MMP cleavage sites in the 600 –710 PTK7 sequenceregion as predicted by the positional weight matrix approachThe high score directly correlates with the high cleavage probability. PWM, posi-tional weight matrix.

Cleavage site Residues PWM score Mutant site PWM score

PKP2LI 621–622 4.463 PKP2DI �1.32PRM2HI 641–642 3.006 PRR2HI �2.08PYK2MI 700–701 2.731 PYK2DI �1.14




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levels might be inversely correlated with tumor aggressivenessand metastatic potential.MT1-MMP/PTK7 Axis in the Zebrafish Embryogenesis—To

confirm whether MT1-MMP regulates Wnt/PCP in diverseorganisms, we used the zebrafish (Danio rerio) developmentalmodel to analyze the role of the MT1-MMP/PTK7 axis in reg-ulating CE movements, a non-canonical Wnt/PCP-dependentprocess, in embryos. PTK7 is reportedly required for PCP andCE in mouse and frog development (15, 16). Studies of non-canonical Wnt/PCP signaling in the zebrafish model are alsowell established (26, 65). Zebrafish PTK7 is already expressed atan early, 6 h postfertilization, stage of embryogenesis, and,therefore, it is present at the right time during gastrulation togovern CE (Fig. 6A). To evaluate whether human and zebrafishproteins could be similarly proteolytically processed, wesequenced zebrafish PTK7 cDNA (deposited in GenBankTM;accession numberGU211905) and compared it with the humangene. We observed a high level of sequence homology betweenthe zebrafish and human genes, including in the region encod-ing the human PKP6212LI cleavage site (Fig. 6C), suggestingthat PTK7 could be cleaved by MT1-MMP in both species.Usingmorpholino knockdown approaches, we observed that

PTK7 silencing (Fig. 6B) induced characteristic CE abnormali-ties in zebrafish embryos. These characteristic abnormalitiesincluded a short anterior-posterior body axis and a wide lateralaxis in the zebrafish (Fig. 7, A and C). Treatment of embryoswith theMMP hydroxamate inhibitors (GM6001 and AG3340)induced similar CE phenotypes (Fig. 7A) accompanied by accu-

mulation of full-length PTK7 in the 2–3-day embryos (Fig. 7B).The developmental defects caused by GM6001 and AG3340were similar to PCP and CE phenotypes reported by othersfollowing silencing of MT1-MMP in zebrafish (27). We alsoobserved thatMT1-MMP and PTK7 interact genetically; injec-tions of low, subthreshold dosages of MT1-MMP and PTK7morpholinos together caused a synergistic effect on the CEphenotype (Fig. 7D).

DISCUSSION

The importance of receptor shedding by MT1-MMP is welldocumented in cancer (56, 66–73). Our proteomics studiesidentified cellular PTK7 pseudokinase as a primary cleavagetarget of MT1-MMP and as a link to the Wnt/PCP pathway.Our subsequent studies confirmed that the full-length mem-brane PTK7 is most efficiently targeted by MT1-MMP, espe-

FIGURE 3. MT1-MMP cleaves the PKP6212LI site in the full-length PTK7sequence. A, a structure model of full-length PTK7. Ig domains 1–7 and thetransmembrane and kinase domains are shown in green, black, and red,respectively. Cleavage of the PKP6212LI site (MMP, red arrow) generates theN-terminal, 70-kDa sPTK7 and the C-terminal, 50-kDa membrane-tetheredfragment. Anti-PTK7, -FLAG, and -V5 staining shows localization of the respec-tive epitopes. B, a surface model of the Ig 7 domain. The PKP6212LI site (red)is in the exposed region and is therefore accessible to proteolysis. C, theL622D PTK7 mutant is resistant to MT1-MMP proteolysis in HT1080 cells,which express endogenous MT1-MMP. D, the L622D PTK7 mutant is resistantto MT1-MMP proteolysis in MDA-MB-435-MT1 cells in which MT1-MMP isoverexpressed.

FIGURE 4. PTK7 co-localizes with MT1-MMP in cells. A, endogenous PTK7(red) is localized at cell-cell junctions (arrowheads) in normal 184B5 mammarycells. Endogenous MT1-MMP (green) is seen at cell-cell junctions and at thecell leading edge (arrows). B, endogenous PTK7 immunoreactivity is reducedat cell-cell junctions in MDCK-MT1 cells. C, in MDCK-MT1 cells, levels of PTK7with a C-terminal V5 tag are reduced at cell-cell junctions (arrows). The C-ter-minal fragment of PTK7 (anti-V5 staining) but not full-length PTK7 (anti-PTK7staining) accumulates at the leading edge in migrating MDCK-MT1 cells(arrows). D, PTK7 immunoreactivity co-localizes with MT1-MMP and isreduced in breast cancer tissue relative to normal mammary tissue.




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cially when compared with other receptors, including CD44.Cellular MT1-MMP functions as a principal sheddase of PTK7and directly cleaves the exposed PKP6212LI sequence of theseventh Ig-like domain of the full-length membrane PTK7.MT1-MMP proteolysis generates the C-terminal, membrane-tethered (50-kDa) and the N-terminal, soluble (70-kDa) frag-ments of PTK7. In turn, inactivation of the cleavage site gener-

ates the uncleavable PTK7 mutant (L622D) that is resistant toMT1-MMP proteolysis.Because PTK7 is an essential component of the Wnt/PCP

pathway, we analyzed the effect of the full-length membranePTK7 and sPTK7 on the actin cytoskeleton, a downstream tar-get of the Wnt/PCP signaling. The precise molecular mecha-nism of PTK7 signaling leading to the regulation of the actomy-osin contraction is not yet understood; however, eitherDishevelled (Dsh) or plexins may be interacting partners ofPTK7 (14, 74, 75).The availability of the cells transfected with the full-length,

soluble, and uncleavable L622D PTK7 constructs allowed us todemonstrate that PTK7 affects the downstream events of theWnt/PCP pathway and that the membrane full-length PTK7and especially the uncleavable L622D mutant reorganize theactin cytoskeleton, repress the MLC phosphorylation, alter theactomyosin contraction, and inhibit cancer cell invasion.MT1-MMP silencing recapitulates the effects that are observed in thecells with the enforced expression of the L622D PTK7 mutant.Consistently, MT1-MMP proteolysis reverses the effects of thefull-length PTK7 on cell functions.The enforced expression of sPTK7 inHT1080 cells, however,

significantly up-regulated RhoA activation, the upstream eventof the Wnt/PCP pathway, rather than the downstream pMLCand actin reorganization. It is likely that the high preexistinglevels of pMLC in the highly migratory HT1080 cells make anyfurther increase of these parameters nearly impossible. As aresult, the expression of MT1-MMP alone does not cause anoticeable effect on RhoA. In agreement, Rho-ROCK-myosinsignaling mediates MT1-MMP-induced cellular aggregation ofkeratinocytes, but the overexpression of MT1-MMP itself doesnot result in a readily detectable increase of RhoA activation(76). MT1-MMP and Rho-ROCK activity and MLC phosphor-ylation were also demonstrated to play an important role inpodosome formation and cell migration (16, 60, 76–78) andembryogenesis (26, 27, 79). Conversely, MT1-MMP silencingaffected pMLC more noticeably, especially if compared withRhoA. Evidently, the effects of the multifunctional MT1-MMPon the net levels of pMLC and RhoA in the highly migratoryHT1080 cells are more complex (80) than the MT1-MMP/PTK7 interactions alone and involve multiple parameters thatare additional and distinct from PTK7.Because of the presence of the link between PTK7 andMT1-

MMP that we detected in cancer cells, we investigated the roleof the PTK7/MT1-MMP axis in embryogenesis using thezebrafish (D. rerio) developmental model. Our results suggestthat zebrafish PTK7 is already expressed as early as 6 h postfer-tilization and that PTK7 is present at the right time duringgastrulation to govern CE in the zebrafish embryo. Both tran-scriptional silencing of PTK7 and inhibition of MT1-MMPactivity, either by small molecule inhibitors or by transcrip-tional silencing, led to characteristic CE abnormalities, includ-ing a short anterior-posterior body axis (dwarfism) and a widelateral axis in the zebrafish. In agreement, subthreshold dosagesof MT1-MMP and PTK7 morpholinos together caused a syn-ergistic effect on the CE phenotype and the developmentalabnormalities in zebrafish.

FIGURE 5. PTK7 regulates the cytoskeleton and cell invasion. A, HT1080cells transiently transfected with full-length PTK7 (green; asterisk indicatesPTK7-expressing cells) rearrange the actin cytoskeleton (red). DNA, DAPInuclear staining. B, phalloidin staining of the actin cytoskeleton in HT1080cells transfected with the indicated PTK7 constructs. C, pMLC, activatedRhoA (GTP-RhoA), MT1-MMP, and �-actin (a loading control). D, collagengel contraction. E, cell invasion through a type 1 collagen matrix in HT1080cells transfected with the indicated MT1-MMP and PTK7 constructs. FBS(10%) was used as a chemoattractant. The dotted line shows cell invasionwithout chemoattractant in serum-free medium (SF). *, p � 0.05 whencompared with HT1080 cells in the presence of FBS. To facilitate the directcomparison of the resulting pMLC bands, equal amounts of the total pro-tein (50 �g/lane) were analyzed in C. Similarly, equal amounts (1 mg oftotal protein) were used in the GTP-RhoA pull-down experiments shownin C. Error bars, S.D.




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Regardless of the opposing effect of PTK7 morpholino andsmall molecule MMP inhibitors on the levels of the full-lengthmembrane PTK7, these treatments resulted in similar CEdefects in the zebrafish embryos. It appears that both in cancercells and normal embryogenesis, not the level of membranePTK7 alone but also the well balanced ratio of the full-lengthPTK7 to sPTK7plays an important role in regulating the overalleffect of this pseudokinase on cell functions. In agreement,according to the findings of others, both overexpression and

silencing of MT1-MMP resulted ina similar CE phenotype in zebrafish(27, 45). Taken together, our datasuggest that the MT1-MMP/PTK7axis plays an important role in nor-mal embryogenesis in the course ofgastrulation in zebrafish.Our data in zebrafish correlate

well with the role PTK7 plays inpolarized cell motility and CE dur-ing mouse gastrulation (15). Thus,in embryos mutant for PTK7, theCE is severely affected. Althoughthere is no alternative splicing in themurine PTK7 gene (18), the pres-ence of the full-length, 140-kDaPTK7 and a PTK7 fragment that issimilar to the sPTK7 species wasreported in mice (16). Most excit-ingly, very recently an N-ethyl-N-nitrosourea-induced mutant,named chuzhoi (chz), has beenreported in mice (44). The chzmutation resulted in the insertion ofthree amino acids (Ala-Asn-Pro)into the junction region between thefifth and the sixth Ig-like domains ofPTK7. The Ala-Asn-Pro insertiondid not change themembrane PTK7levels in mice but led to the degra-dation of the sPTK7 species. Theresulting imbalance between mem-brane PTK7 and sPTK7 led to char-acteristics consistent with defectiveCE, including a shortened body axisand multiple defects in heart, lung,and inner ear development. Theseobservations agree very well withour results thatwe generated in can-cer cells and zebrafish.As a result, we believe that a fine

balance between the protease andPTK7 is required for normalembryo development. In general, itis now highly likely that the MT1-MMP/PTK7 axis plays an essentialrole in embryogenesis in the courseof gastrulation in vertebrates. Con-versely, aberrations of the MT1-

MMP/PTK7 axis seem to be the cause of abnormal CE duringgastrulation in vertebrates.Conclusions—Overall, we established that the full-length

PTK7 down-regulates myosin light chain phosphorylation,actin cytoskeleton organization, and actomyosin contraction(all downstream events in the Wnt/PCP pathway) and that itstrongly inhibits cell invasion. PTK7 is a major cleavage targetof MT1-MMP in the plasma membrane. MT1-MMP directlycleaves the PKP6212LI sequence in an exposed region of PTK7,

FIGURE 6. PTK7 expression in zebrafish embryos. A, RT-PCR analysis of PTK7 in embryo extracts using twoselective primer pairs. beta-actin, loading control. B, treatment with specific morpholinos silenced PTK7 expres-sion in zebrafish. Equal amount of total protein (50 �g) was loaded per lane. C, alignment of human andzebrafish PTK7 sequence. The MT1-MMP cleavage site (PKP6212LI) is underlined in the human sequence.

FIGURE 7. MT1-MMP and PTK7 interact in zebrafish embryogenesis. A, MMP hydroxamate inhibitors orPTK7 morpholino (PTK MO) induce a CE phenotype in zebrafish. Day 5 embryos are shown. B, GM6001 causesaccumulation of full-length PTK7 in embryos. An equal amount of total protein (50 �g) was loaded per lane. C,PTK7 regulates CE in zebrafish. Control and PTK7 morpholino embryos (10 h postfertilization) were stained witha myoD RNA probe to identify mesodermal tissues. The distance between bilateral adaxial cells is presented asa percentage relative to the intact control. D, synergistic effect of MT1-MMP and PTK7 in embryo development.Embryos received low doses of MT1-MMP (MMP-14a�b) and PTK7 morpholinos (2 ng/embryo) on Day 0. Day3 embryos are shown. Error bars, S.D.




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generating the N-terminal, soluble PTK7 ectodomain. The lat-ter forms a complex with the full-length membrane PTK7.MT1-MMP proteolysis reverses the inhibitory action of thefull-length PTK on cell locomotion. MT1-MMP silencing andthe analysis of the uncleavable L622D PTK7 mutant also con-firm the significance of MT1-MMP proteolysis of PTK7 in cellfunctions. Our novel data suggest that the MT1-MMP/PTK7axis plays an important role in the regulation of the non-canon-ical Wnt/PCP pathway and polarized cell motility both inmalignancy and vertebrate embryo development. Our resultsbring us a step closer to the development of selective therapeu-tics to target the MT1-MMP/PTK7 axis and the Wnt/PCPpathway in a clinically beneficial manner.REFERENCES1. Gao, C., and Chen, Y. G. (2010) Cell. Signal. 22, 717–7272. Schlessinger, K., Hall, A., andTolwinski, N. (2009)GenesDev.23, 265–2773. Simons, M., and Mlodzik, M. (2008) Annu. Rev. Genet. 42, 517–5404. Katoh, M. (2005) Oncol. Rep. 14, 1583–15885. Jessen, J. R. (2009) Zebrafish 6, 21–286. Wang, Y. (2009)Mol. Cancer Ther. 8, 2103–21097. Vladar, E. K., Antic, D., and Axelrod, J. D. (2009) Cold Spring Harb. Per-

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Alex Y. StronginandAndrei V. Chernov, Wenhong Zhu, Ilian A. Radichev, Danhua Zhang, P. Duc Dong

Vladislav S. Golubkov, Alexei V. Chekanov, Piotr Cieplak, Alexander E. Aleshin,IMPLICATIONS IN CANCER AND EMBRYOGENESIS

Efficient Proteolytic Target of Membrane Type-1 Matrix Metalloproteinase: The Wnt/Planar Cell Polarity Protein-tyrosine Kinase-7 (PTK7) Is a Highly

doi: 10.1074/jbc.M110.165159 originally published online September 13, 20102010, 285:35740-35749.J. Biol. Chem.

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