The Wnt/planar cell polarity protein-tyrosine kinase-7 (PTK7) is a highly efficient proteolytic target of membrane type-1 matrix metalloproteinase: Implications in cancer and embryogenesis

Article (PDF Available)inJournal of Biological Chemistry 285(46):35740-9 · November 2010with60 Reads
DOI: 10.1074/jbc.M110.165159 · Source: PubMed
Abstract
PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.
The Wnt/Planar Cell Polarity Protein-tyrosine Kinase-7 (PTK7)
Is a Highly Efficient Proteolytic Target of Membrane Type-1
Matrix Metalloproteinase
IMPLICATIONS 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. Strongin
1
From the Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037
PTK7 is an essential component of the Wnt/planar cell polar-
ity (PCP) pathway. We provide evidence that the Wnt/PCP
pathway converges with pericellular proteolysis in both normal
development and cancer. Here, we demonstrate that membrane
type-1 matrix metalloproteinase (MT1-MMP), a key proinva-
sive proteinase, functions as a principal sheddase of PTK7.
MT1-MMP directly cleaves the exposed PKP
621
2LI sequence
of the seventh Ig-like domain of the full-length membrane PTK7
and generates, as a result, an N-terminal, soluble PTK7 frag-
ment (sPTK7). The enforced expression of membrane PTK7 in
cancer cells leads to the actin cytoskeleton reorganization and
the inhibition of cell invasion. MT1-MMP silencing and the
analysis of the uncleavable L622D PTK7 mutant confirm the
significance of MT1-MMP proteolysis of PTK7 in cell functions.
Our data also demonstrate that a fine balance between the met-
alloproteinase activity and PTK7 levels is required for normal
development of zebrafish (Danio rerio). Aberration of this bal-
ance by the proteinase inhibition or PTK7 silencing results in
the PCP-dependent convergent extension defects in the
zebrafish. Overall, our data suggest that the MT1-MMP-PTK7
axis plays an important role in both cancer cell invasion and
normal embryogenesis in vertebrates. Further insight into these
novel mechanisms may promote understanding of directional
cell motility and lead to the identification of therapeutics to
treat 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 and well conserved func-
tion of the non-canonical pathway is to control PCP
2
and direc-
tional cell motility (8). PCP governs the orientation of cells in a
monolayer of a tissue plane (front-back orientation) in such a
way that all cells within the monolayer are aligned in the same
direction. As a result, PCP is important for the directed collec-
tive cell movements and orchestrates the synchronized cell
arrangements within the tissue plane in the course of a plethora
of biological processes (2, 4, 6–11).
The first PCP signaling events occur at a gastrulation stage of
embryogenesis to regulate the polarized cell movement and
accomplish convergent extension (CE) for the anterior-poste-
rior body axis elongation, neural tube closure, and craniofacial
morphogenesis (8, 9, 11, 12). CE failure results in the multiple
severe developmental defects, including a shortened body axis
(dwarfism), defective neural system, and craniofacial abnor-
malities. Defects in the non-canonical Wnt/PCP pathway are
linked 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 promote
cancer cell motility and invasion (6, 13). Evidently, an in depth
mechanistic understanding of the PCP mechanism and 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). The
full-length membrane PTK7 receptor consists of seven extra-
cellular Ig domains, a transmembrane region, and a catalytically
inert cytoplasmic tyrosine kinase (PTK) domain (17–20). PTK7
is evolutionary conserved, and its orthologs include mouse
PTK7, chicken KLG, Drosophila Dtrk/Off-track (OTK), and
Hydra Lemon (21). PTK7 mutant mice that expressed a 1–114
PTK7 truncation died perinatally with severe defects in neural
tube closure, a CE process (16). Overexpression of the mutant
PTK7 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). The
expression 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 the MMP family, is a
major mediator of pericellular proteolytic events in cancer cells
(28). MT1-MMP cleaves ECM proteins, initiates activation of
soluble MMPs, and controls the functionality of cell adhesion
and signaling receptors. MT1-MMP is a prototypic member of
* This work was supported, in whole or in part, by National Institutes of Health
Grants CA83017 and CA77470 (to A. Y. S.).
The nucleotide sequence(s) reported in this paper has been submitted to the Gen-
Bank
TM
/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: strongin@burnham.org.
2
The abbreviations used are: PCP, planar cell polarity; CE, convergent exten-
sion; MLC, myosin light chain; pMLC, phosphorylated MLC; MMP, matrix
metalloproteinase; 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-Darby
canine 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 distinguished
from soluble MMPs by a C-terminal transmembrane domain
and a cytoplasmic tail (29–31). MT1-MMP is synthesized as a
latent zymogen that requires proteolytic processing of the
N-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 soluble MMPs, is ideally posi-
tioned to regulate pericellular proteolysis and the functionality
of cell receptors (36). In migrating cells, MT1-MMP accumu-
lates predominantly at the leading and trailing edges and, as a
result, contributes most efficiently to cell locomotion (37–39).
Knock-out of MT1-MMP has the most significant phenotype
among MMP gene knock-out mice; MT1-MMP knock-out
mice are dwarfs and die at adulthood (40, 41). Likewise, a loss of
the structurally similar primordial At2-MMP induces dwarfism
in 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 morphogenesis
in zebrafish, suggesting that a stringent control of MT1-MMP
activity is essential in normal development (27, 44, 45). The molec-
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 human
and 21,036 mouse genes from the COEXPRESdb database indi-
cate that MT1-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 cleaves
the full-length membrane PTK7, that this cleavage generates
the sPTK7 species, and, most importantly, that this proteolytic
event is ubiquitous in multiple cell systems. Taken together, our
experimental data suggest that the pericellular proteolysis and
PCP mechanisms converge in the regulation of directional cell
migration and that they work in concert in processes as diverse
as 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 of
Dr. Xiaowei Lu (University of Virginia, Charlottesville, VA). A
goat polyclonal antibody (catalog no. AF4499) against the
N-terminal 31–199 portion of PTK7 was from R&D. A murine
monoclonal 3G4 antibody (catalog no. MAB1767) against the
catalytic domain of MT1-MMP, a rabbit AB8104 antibody to
the hinge domain of MT1-MMP, and the GM6001 hydroxam-
ate inhibitor were from Chemicon. A murine monoclonal anti-
body to the V5 tag was from Invitrogen. A murine monoclonal
FLAG M2 antibody, the FLAG M2 antibody-agarose beads, and
a polyclonal rabbit TGN46 antibody (catalog no. T7576) were
from Sigma. The phosphomyosin light chain 2 (Ser
19
) rabbit
polyclonal antibody (catalog no. 3671) was from Cell Signaling.
Rhotekin-RBD agarose beads and a RhoA monoclonal antibody
(catalog no. ARH01) were from Cytoskeleton. EZ-Link sulfos-
uccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate was
from Pierce. Human fibrosarcoma HT1080, breast carcinoma
MCF7, mammary epithelial MCF10A and 184B5 cells, and
Madin-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 184B5
cells were grown in mammary epithelial growth medium
(Lonza). Other cell lines were grown in DMEM supplemented
with 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-length
MT1-MMP (MCF7-MT1, HT1080-MT1, 184B5-MT1, MDA-
MB-435-MT1, and MDCK-MT1 cells, respectively) were
established and characterized earlier (47, 48). HT1080 cells
with the over 90% transcriptional silencing of MT1-MMP and
the required scrambled controls were obtained and extensively
characterized earlier (47, 49, 50). The full-length wild-type
PTK7 cDNA (OriGene) was amplified by the PCR using the
selective PTK7 primers. The construct was subcloned into the
pcDNA3.1D/V5-His-TOPO directional TOPO expression vec-
tor (Invitrogen). Where indicated, the PTK7 construct was
C-terminally tagged with the V5-His and FLAG tags. The full-
length PTK7-FLAG template was used to generate the L622D,
M641R, and M701D mutants 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 cells
using Lipofectamine 2000 (Invitrogen). The full-length and the
TABLE 1
Oligonucleotide 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-GGAAGATGTGCCGCCTGGGTCCCAGCTTGG
M701D 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 and MDCK-MT1-sPTK7 cells, respectively. HT1080 sta-
bly transfected with MT6-MMP were used as an additional
control in our study (51).
Cell Surface Biotinylation, Two-dimensional PAGE, and Pro-
tein Identification by LC/MS/MS —Cell surface proteins were
biotinylated by incubating cells for1honiceinPBScontaining
0.1 mg/ml EZ-Link sulfosuccinimidyl 2-(biotinamido)-ethyl-
1,3-dithiopropionate. Cells were lysed in 20 m
M Tris-HCl, 150
m
M NaCl, 1% deoxycholate, 1% IGEPAL, pH 7.4, supplemented
with a protease inhibitor mixture set III (Sigma), 1 m
M phenyl-
methylsulfonyl fluoride, and 10 m
M EDTA. Biotinylated pro-
teins were precipitated from cell lysates with streptavidin-aga-
rose beads (Sigma). Biotinylated proteins were eluted from the
beads using 50 m
M DTT. The samples were then alkylated using
a ReadyPrep reduction-alkylation kit (Bio-Rad) and separated
by two-dimensional PAGE using a PROTEAN II xi Cell (Bio-
Rad). The gels were stained with SimplyBlue SafeStain (Invitro-
gen). The gel images were analyzed with ImageJ software
(National Institutes of Health). The individual stained spots
were 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 LTQ
XL linear ion trap mass spectrometer (Thermo Scientific).
MS/MS spectra were searched against the Swiss-Prot data base
using 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 10
6
) were
captured on streptavidin-agarose beads (20
l of a 50% slurry)
and co-incubated for the indicated time at 37 °C with the
recombinant catalytic domain of MT1-MMP (20 n
M)in100
l
of 50 m
M HEPES, pH 6.8, containing 10 mM CaCl
2
, 0.5 mM
MgCl
2
, and 50
M ZnCl
2
. 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 on
the known structures of the homologues using the program
MODWEB (52, 53). The first six Ig domains of PTK7 (residues
28–588) 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 entry
2DM7 (Kiaa1556, residues 22–105, sequence identity 38%).
The transmembrane region of PTK7 (residues 703–778) was
built using Protein Data Bank entry 1SYS (Hla), and the PTK7
kinase domain (residues 789–1072) was built using a fragment
of Protein Data Bank entry 2BDF (Src, residues 258 –525,
sequence identity 38%). The modeled fragments of PTK7 were
merged together and visualized by using PyMOL software
(DeLano Scientific).
Immunofluorescence—Cells grown on a glass coverslip were
fixed with 4% paraformaldehyde for 10 min, permeabilized with
0.1% Triton X-100 for 5 min, and then blocked in 1% casein for
1 h. Cells were stained with the primary antibodies (dilution
1:1,000) for 16 h at 4 °C, followed by staining with the secondary
antibodies conjugated with AlexaFluor 488 or AlexaFluor 594
(Molecular Probes; dilution 1:500) for2hatambient tempera-
ture. The coverslips were mounted in the Vectashield mount-
ing medium with DAPI (Vector Laboratories). Images were
acquired on an Olympus BX51 fluorescence microscope
equipped with a MagnaFire digital camera and MagnaFire 2.1C
software (Olympus).
Rho Activation Assay—The Rho activation assay kit (Cy-
toskeleton) was used to estimate the cellular GTP-bound RhoA.
Briefly, the active, GTP-bound RhoA was precipitated from the
cell lysate using rhotekin-RBD beads. The precipitated RhoA
was analyzed by Western blotting with a RhoA monoclonal
antibody.
Collagen Gel Contraction Assay—Cells (1 10
5
) were mixed
with 0.2 ml of type I collagen in DMEM (0.3 mg/ml) on ice and
placed in wells of a 24-well low adhesion cell culture plate. After
gel polymerization at 37 °C for 30 min, the growth medium (0.5
ml) was added to the wells. Following a 24-h incubation, the gel
images 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 with
type I collagen (2.5
g/well; BD Bioscience). Cells (1 10
5
/
well) were placed in serum-free DMEM (0.1 ml) into the upper
chamber. The 10% FBS-containing DMEM (used as a chemoat-
tractant, 0.6 ml) was placed in the lower chamber. Serum-free
DMEM (0.6 ml) was used as a control. Cells were allowed to
invade 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 upper
membrane surface were removed with a cotton swab. The dye
from the cells that migrated onto the membrane’s lower surface
was extracted with 1% SDS (0.25 ml). The resulting A
570 nm
was
measured using a SpectraFluor Plus plate reader (Tecan). The
assays were run in triplicate in three independent experiments.
Immunohistochemistry—Breast cancer tissue arrays BR962
were from US Biomax. After deparaffinization and antigen
retrieval, the arrays were stained using the goat PTK7 poly-
clonal antibody and the MT1-MMP 3G4 monoclonal antibody,
followed by the staining with the secondary antibodies conju-
gated with AlexaFluor 488 or AlexaFluor 594.
Zebrafish Maintenance, Treatment with Inhibitors and Mor-
pholino Injection—The zebrafish (AB strain) were maintained
under the standard laboratory conditions at 28.5 °C. One-cell
stage embryos were collected from natural matings. GM6001
(100
M) was added to the one-cell stage embryos and replaced
twice daily. MT1-MMP (MMP14a and MMP14b) and PTK7
morpholinos 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 embryos were lysed in 20 m
M Tris-HCl, 150
m
M NaCl, 1% deoxycholate, 1% IGEPAL, pH 7.4, supplemented
with a protease inhibitor mixture set III (Sigma), 1 m
M phenyl-
methylsulfonyl fluoride, and 10 m
M EDTA. The lysates were
analyzed 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 RNA probe myoD
Proteolysis and Non-canonical Wnt Signaling
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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 RNA
samples were extracted from the lysates and then purified using
the RNA miniprep columns (Zymo Research). First-strand
cDNA was synthesized using the purified RNA samples (1
g),
SuperScript II reverse transcriptase (Invitrogen), and a random
primer (100 ng). The forward and reverse primers for the
PCR 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. The
amplification reactions (25
l) included the cDNA (50 ng)
and the respective primers (0.6
M). PCRs (30 cycles) were
performed using denaturation at 95 °C for 30 s, annealing at
58 °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 gel
extraction kit (Qiagen). Both the sense and antisense cDNA
strands were sequenced to obtain the nucleotide sequence of
PTK7. As a result, the 1782-bp cDNA sequence of zebrafish
PTK7 was assembled and deposited in GenBank
TM
(acces
-
sion number GU211905).
RESULTS
PTK7 Is a Proteolytic Target of MT1-MMP—To search for
novel cell surface cleavage targets of MT1-MMP, we compared
two-dimensional gel profiles of biotin-labeled plasma mem-
brane proteins from normal mammary 184B5 epithelial cells
with those from 184B5 cells stably transfected with MT1-MMP
(184B5-MT1 cells) (Fig. 1). The identity of the protein spots was
determined using LC/MS/MS. We readily detected reduced
levels of PTK7 in 184B5-MT1 cells. We then determined PTK7
levels in several normal and cancer cell lines of a diversified
tissue 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 of
full-length membrane PTK7. In contrast, PTK7 levels were low
in highly metastatic, invasive breast carcinoma MDA-MB-435
cells and in 184B5 and MCF7 cells in which MT1-MMP had
been overexpressed (184B5-MT1 and MCF7-MT1 cells, re-
spectively). Treatment of cells with GM6001 (a wide range
hydroxamate MMP inhibitor) or tissue inhibitor of metallopro-
teinases-2 (TIMP-2; a potent MT1-MMP inhibitor) increased
levels of full-length PTK7 in MCF7-MT1 cells to those
observed in MT1-MMP-deficient MCF7 cells. TIMP-1 (an
inefficient MT1-MMP inhibitor) had no effect (Fig. 2B). MT1-
MMP activity was correlated with the presence of sPTK7 in the
medium. Expression of the lipid raft-associated MT6-MMP in
cells did not promote a similar effect (Fig. 2C). sPTK7 was also
detected in plasma membrane samples, suggesting that sPTK7
and 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). Taken
together, our results indicate that PTK7 is a major target of
MT1-MMP.
MT1-MMP Directly Cleaves the PKP
621
2LI Site of PTK7
We next determined the identity of the cleavage site and the
cleavage events that result in sPTK7. To predict PTK7 cleavage
site(s) that could lead to sPTK7 generation, we analyzed the
region encompassing PTK7 amino acids 600 –710 for potential
MMP cleavage sites using software we developed (58) (Table 2).
We identified three potential MT1-MMP cleavage sites
(PKP
621
2LI, PRM
641
2HI, and PYK
700
2MI) in this region.
Corresponding L622D, M641R, and M701D PTK7 point
mutants with inactivated cleavage sites were generated, and
the constructs were expressed in fibrosarcoma HT1080 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 L622D
mutant was fully resistant to MT1-MMP proteolysis in both
HT1080 and MDA-MB-435-MT1 cells. Structural modeling
suggests that the PKP
621
2LI cleavage site is localized in the
exposed region of the seventh Ig-like domain of PTK7, and, as a
result, it is probably accessible to proteolysis (Fig. 3).
MT1-MMP and PTK7 Co-localize in the CellsThe
observed proteolysis of PTK7 by MT1-MMP suggests that the
proteinase 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 by
two-dimensional gel electrophoresis (bottom panels). The densitometry pro-
file of the selected region (square) is shown in the top panels. The identity of
PTK7 (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, which
co-express endogenous MT1-MMP and PTK7. Enforced
expression of MT1-MMP in MDCK epithelial cells (MDCK-
MT1 cells) resulted in reduced levels of full-length membrane
PTK7 both at cell-cell junctions and at the leading edge of
migrating 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 migrating
MDCK-MT1 cells. These observations indicate the enhanced
proteolysis 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 of
membrane PTK7 in migrating cells. Our findings support the
role of PTK7 in cell migration and correlate with earlier obser-
vations by others who showed that PTK7 contributes to neural
crest migration in Xenopus by
recruiting Dishevelled (14). In
agreement, Van Gogh-like 2,
another important regulator of the
non-canonical Wnt pathway, also
co-localizes with MT1-MMP and
redistributes toward the leading
edge of the polarized human cancer
cells (27).
Previous reports indicate that
PTK7 levels are inversely correlated
with melanoma tumorigenicity
(25). To further investigate the rela-
tionship between PTK7 and cancer,
we analyzed PTK7 in breast cancer
biopsies by immunostaining and
observed reduced membrane PTK7
immunoreactivity in tumor lesions
compared with normal mammary
tissue. 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 and
Cell Invasion—Although previous
reports indicate that PTK7 regu-
lates PCP, the precise role of PTK7
in the non-canonical Wnt/PCP
signaling 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 HT1080
cells transfected with MT1-MMP siRNA and PTK7 expression
constructs. 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 pMLC
compared with those seen in the parental HT1080 cells (Fig. 5,
A and B). Similarly, silencing of MT1-MMP using siRNA
decreased pMLC levels. Conversely, expression of sPTK7 alone
dramatically stimulated RhoA activity in cells (Fig. 5C).
MLC phosphorylation is critical for actomyosin contractility,
which, in turn, correlates with the ability of cells to contract
three-dimensional collagen gels (60 62). In agreement, pMLC
levels 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 actin
cytoskeleton dynamics are known to be associated with cell
invasion (63, 64), we next analyzed the invasive capacity of
HT1080 cells that had been transfected with MT1-MMP
siRNA and PTK7 constructs. Cells transfected with full-length
PTK7 and, particularly, with the uncleavable L622D construct
were significantly less invasive compared with HT1080 control
cells, whereas overexpression of sPTK7 did not promote any
significant effect. Conversely, PTK7 proteolysis by MT1-MMP
reversed 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-invasive
breast 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 and
TIMP-2 (but not TIMP-1) inhibit MT1-MMP proteolysis of membrane PTK7. Biotin-labeled plasma membrane
proteins from MCF7 and MCF7-MT1 cells were analyzed by immunoblotting with the PTK7 antibody. C, sPTK7
is 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 membrane
proteins from MCF7 cells were co-incubated with the recombinant catalytic domain of MT1-MMP. Samples
were 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 and
MT1-MMP constructs (MDCK-MT1-PTK7 cells) and MDCK cells that co-express MT1-MMP with the soluble PTK7
construct (MDCK-MT1-sPTK7 cells). Where indicated, GM6001 was added to the cells.
TABLE 2
Potential MT1-MMP cleavage sites in the 600 –710 PTK7 sequence
region as predicted by the positional weight matrix approach
The 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 1.32
PRM2HI 641–642 3.006 PRR2HI 2.08
PYK2MI 700–701 2.731 PYK2DI 1.14
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levels might be inversely correlated with tumor aggressiveness
and metastatic potential.
MT1-MMP/PTK7 Axis in the Zebrafish Embryogenesis—To
confirm whether MT1-MMP regulates Wnt/PCP in diverse
organisms, we used the zebrafish (Danio rerio) developmental
model to analyze the role of the MT1-MMP/PTK7 axis in reg-
ulating CE movements, a non-canonical Wnt/PCP-dependent
process, in embryos. PTK7 is reportedly required for PCP and
CE in mouse and frog development (15, 16). Studies of non-
canonical Wnt/PCP signaling in the zebrafish model are also
well established (26, 65). Zebrafish PTK7 is already expressed at
an early, 6 h postfertilization, stage of embryogenesis, and,
therefore, it is present at the right time during gastrulation to
govern CE (Fig. 6A). To evaluate whether human and zebrafish
proteins could be similarly proteolytically processed, we
sequenced zebrafish PTK7 cDNA (deposited in GenBank
TM
;
accession number GU211905) and compared it with the human
gene. We observed a high level of sequence homology between
the zebrafish and human genes, including in the region encod-
ing the human PKP
621
2LI cleavage site (Fig. 6C), suggesting
that PTK7 could be cleaved by MT1-MMP in both species.
Using morpholino knockdown approaches, we observed that
PTK7 silencing (Fig. 6B) induced characteristic CE abnormali-
ties in zebrafish embryos. These characteristic abnormalities
included a short anterior-posterior body axis and a wide lateral
axis in the zebrafish (Fig. 7, A and C). Treatment of embryos
with the MMP 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 AG3340
were similar to PCP and CE phenotypes reported by others
following silencing of MT1-MMP in zebrafish (27). We also
observed that MT1-MMP and PTK7 interact genetically; injec-
tions of low, subthreshold dosages of MT1-MMP and PTK7
morpholinos together caused a synergistic effect on the CE
phenotype (Fig. 7D).
DISCUSSION
The importance of receptor shedding by MT1-MMP is well
documented in cancer (56, 66–73). Our proteomics studies
identified cellular PTK7 pseudokinase as a primary cleavage
target 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 PKP
621
2LI site in the full-length PTK7
sequence. A, a structure model of full-length PTK7. Ig domains 1–7 and the
transmembrane and kinase domains are shown in green, black, and red,
respectively. Cleavage of the PKP
621
2LI site (MMP, red arrow) generates the
N-terminal, 70-kDa sPTK7 and the C-terminal, 50-kDa membrane-tethered
fragment. Anti-PTK7, -FLAG, and -V5 staining shows localization of the respec-
tive epitopes. B, a surface model of the Ig 7 domain. The PKP
621
2LI site (red)
is in the exposed region and is therefore accessible to proteolysis. C, the
L622D PTK7 mutant is resistant to MT1-MMP proteolysis in HT1080 cells,
which express endogenous MT1-MMP. D, the L622D PTK7 mutant is resistant
to MT1-MMP proteolysis in MDA-MB-435-MT1 cells in which MT1-MMP is
overexpressed.
FIGURE 4. PTK7 co-localizes with MT1-MMP in cells. A, endogenous PTK7
(red) is localized at cell-cell junctions (arrowheads) in normal 184B5 mammary
cells. Endogenous MT1-MMP (green) is seen at cell-cell junctions and at the
cell leading edge (arrows). B, endogenous PTK7 immunoreactivity is reduced
at cell-cell junctions in MDCK-MT1 cells. C, in MDCK-MT1 cells, levels of PTK7
with 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-PTK7
staining) accumulates at the leading edge in migrating MDCK-MT1 cells
(arrows). D, PTK7 immunoreactivity co-localizes with MT1-MMP and is
reduced 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 PTK7
and directly cleaves the exposed PKP
621
2LI sequence of the
seventh 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 to
MT1-MMP proteolysis.
Because PTK7 is an essential component of the Wnt/PCP
pathway, we analyzed the effect of the full-length membrane
PTK7 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, either
Dishevelled (Dsh) or plexins may be interacting partners of
PTK7 (14, 74, 75).
The availability of the cells transfected with the full-length,
soluble, and uncleavable L622D PTK7 constructs allowed us to
demonstrate that PTK7 affects the downstream events of the
Wnt/PCP pathway and that the membrane full-length PTK7
and especially the uncleavable L622D mutant reorganize the
actin cytoskeleton, repress the MLC phosphorylation, alter the
actomyosin contraction, and inhibit cancer cell invasion. MT1-
MMP silencing recapitulates the effects that are observed in the
cells with the enforced expression of the L622D PTK7 mutant.
Consistently, MT1-MMP proteolysis reverses the effects of the
full-length PTK7 on cell functions.
The enforced expression of sPTK7 in HT1080 cells, however,
significantly up-regulated RhoA activation, the upstream event
of the Wnt/PCP pathway, rather than the downstream pMLC
and actin reorganization. It is likely that the high preexisting
levels of pMLC in the highly migratory HT1080 cells make any
further increase of these parameters nearly impossible. As a
result, the expression of MT1-MMP alone does not cause a
noticeable effect on RhoA. In agreement, Rho-ROCK-myosin
signaling mediates MT1-MMP-induced cellular aggregation of
keratinocytes, but the overexpression of MT1-MMP itself does
not 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 in
podosome formation and cell migration (16, 60, 76 –78) and
embryogenesis (26, 27, 79). Conversely, MT1-MMP silencing
affected pMLC more noticeably, especially if compared with
RhoA. Evidently, the effects of the multifunctional MT1-MMP
on the net levels of pMLC and RhoA in the highly migratory
HT1080 cells are more complex (80) than the MT1-MMP/
PTK7 interactions alone and involve multiple parameters that
are additional and distinct from PTK7.
Because of the presence of the link between PTK7 and MT1-
MMP that we detected in cancer cells, we investigated the role
of the PTK7/MT1-MMP axis in embryogenesis using the
zebrafish (D. rerio) developmental model. Our results suggest
that zebrafish PTK7 is already expressed as early as 6 h postfer-
tilization and that PTK7 is present at the right time during
gastrulation to govern CE in the zebrafish embryo. Both tran-
scriptional silencing of PTK7 and inhibition of MT1-MMP
activity, 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 wide
lateral axis in the zebrafish. In agreement, subthreshold dosages
of MT1-MMP and PTK7 morpholinos together caused a syn-
ergistic effect on the CE phenotype and the developmental
abnormalities in zebrafish.
FIGURE 5. PTK7 regulates the cytoskeleton and cell invasion. A, HT1080
cells transiently transfected with full-length PTK7 (green; asterisk indicates
PTK7-expressing cells) rearrange the actin cytoskeleton (red). DNA, DAPI
nuclear staining. B, phalloidin staining of the actin cytoskeleton in HT1080
cells transfected with the indicated PTK7 constructs. C, pMLC, activated
RhoA (GTP-RhoA), MT1-MMP, and
-actin (a loading control). D, collagen
gel contraction. E, cell invasion through a type 1 collagen matrix in HT1080
cells transfected with the indicated MT1-MMP and PTK7 constructs. FBS
(10%) was used as a chemoattractant. The dotted line shows cell invasion
without chemoattractant in serum-free medium (SF). *, p 0.05 when
compared with HT1080 cells in the presence of FBS. To facilitate the direct
comparison of the resulting pMLC bands, equal amounts of the total pro-
tein (50
g/lane) were analyzed in C. Similarly, equal amounts (1 mg of
total protein) were used in the GTP-RhoA pull-down experiments shown
in C. Error bars, S.D.
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Regardless of the opposing effect of PTK7 morpholino and
small molecule MMP inhibitors on the levels of the full-length
membrane PTK7, these treatments resulted in similar CE
defects in the zebrafish embryos. It appears that both in cancer
cells and normal embryogenesis, not the level of membrane
PTK7 alone but also the well balanced ratio of the full-length
PTK7 to sPTK7 plays an important role in regulating the overall
effect of this pseudokinase on cell functions. In agreement,
according to the findings of others, both overexpression and
silencing of MT1-MMP resulted in
a similar CE phenotype in zebrafish
(27, 45). Taken together, our data
suggest that the MT1-MMP/PTK7
axis plays an important role in nor-
mal embryogenesis in the course of
gastrulation in zebrafish.
Our data in zebrafish correlate
well with the role PTK7 plays in
polarized cell motility and CE dur-
ing mouse gastrulation (15). Thus,
in embryos mutant for PTK7, the
CE is severely affected. Although
there is no alternative splicing in the
murine PTK7 gene (18), the pres-
ence of the full-length, 140-kDa
PTK7 and a PTK7 fragment that is
similar to the sPTK7 species was
reported in mice (16). Most excit-
ingly, very recently an N-ethyl-
N-nitrosourea-induced mutant,
named chuzhoi (chz), has been
reported in mice (44). The chz
mutation resulted in the insertion of
three amino acids (Ala-Asn-Pro)
into the junction region between the
fifth and the sixth Ig-like domains of
PTK7. The Ala-Asn-Pro insertion
did not change the membrane PTK7
levels in mice but led to the degra-
dation of the sPTK7 species. The
resulting imbalance between mem-
brane PTK7 and sPTK7 led to char-
acteristics consistent with defective
CE, including a shortened body axis
and multiple defects in heart, lung,
and inner ear development. These
observations agree very well with
our results that we generated in can-
cer cells and zebrafish.
As a result, we believe that a fine
balance between the protease and
PTK7 is required for normal
embryo development. In general, it
is now highly likely that the MT1-
MMP/PTK7 axis plays an essential
role in embryogenesis in the course
of gastrulation in vertebrates. Con-
versely, aberrations of the MT1-
MMP/PTK7 axis seem to be the cause of abnormal CE during
gastrulation 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 it
strongly inhibits cell invasion. PTK7 is a major cleavage target
of MT1-MMP in the plasma membrane. MT1-MMP directly
cleaves the PKP
621
2LI sequence in an exposed region of PTK7,
FIGURE 6. PTK7 expression in zebrafish embryos. A, RT-PCR analysis of PTK7 in embryo extracts using two
selective 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 and
zebrafish PTK7 sequence. The MT1-MMP cleavage site (PKP
621
2LI) is underlined in the human sequence.
FIGURE 7. MT1-MMP and PTK7 interact in zebrafish embryogenesis. A, MMP hydroxamate inhibitors or
PTK7 morpholino (PTK MO) induce a CE phenotype in zebrafish. Day 5 embryos are shown. B, GM6001 causes
accumulation 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 with
a myoD RNA probe to identify mesodermal tissues. The distance between bilateral adaxial cells is presented as
a 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-14ab) and PTK7 morpholinos (2 ng/embryo) on Day 0. Day
3 embryos are shown. Error bars, S.D.
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NOVEMBER 12, 2010 VOLUME 285 NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35747
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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 the
full-length PTK on cell locomotion. MT1-MMP silencing and
the analysis of the uncleavable L622D PTK7 mutant also con-
firm the significance of MT1-MMP proteolysis of PTK7 in cell
functions. Our novel data suggest that the MT1-MMP/PTK7
axis plays an important role in the regulation of the non-canon-
ical Wnt/PCP pathway and polarized cell motility both in
malignancy and vertebrate embryo development. Our results
bring us a step closer to the development of selective therapeu-
tics to target the MT1-MMP/PTK7 axis and the Wnt/PCP
pathway in a clinically beneficial manner.
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    • "In addition, much like the invertebrate function of Otk, in vertebrate development, Ptk7 interacts with plexin A1 regulating neural crest migration [70][71][72]. Ptk7 is cleaved by a membrane type matrix metalloprotease (MT1-MMP) affecting its function in both zebrafish and human development, and cancer cell metastasis [61,[73][74][75][76]. Taken together, these findings suggest that Ptk7 is a highly regulated, polarity-determining molecule in a variety of cellular behaviors both during development and in cancers. "
    [Show abstract] [Hide abstract] ABSTRACT: Human development uses a remarkably small number of signal transduction pathways to organize vastly complicated tissues. These pathways are commonly associated with disease in adults if activated inappropriately. One such signaling pathway, Wnt, solves the too few pathways conundrum by having many alternate pathways within the Wnt network. The main or "canonical" Wnt pathway has been studied in great detail, and among its numerous downstream components, several have been identified as drug targets that have led to cancer treatments currently in clinical trials. In contrast, the non-canonical Wnt pathways are less well characterized, and few if any possible drug targets exist to tackle cancers caused by dysregulation of these Wnt offshoots. In this review, we focus on two molecules-Protein Tyrosine Kinase 7 (Ptk7) and Mutated in Colorectal Cancer (Mcc)-that do not fit perfectly into the non-canonical pathways described to date and whose roles in cancer are ill defined. We will summarize work from our laboratories as well as many others revealing unexpected links between these two proteins and Wnt signaling both in cancer progression and during vertebrate and invertebrate embryonic development. We propose that future studies focused on delineating the signaling machinery downstream of Ptk7 and Mcc will provide new, hitherto unanticipated drug targets to combat cancer metastasis.
    Full-text · Article · Jul 2016
    • "Based on our previous results, we have hypothesized that MT1-MMP deactivates the antiinvasive function of the full-length PTK7 (19-21). However, in-depth understanding of these effects was lacking in the earlier studies by us and others (9,17,20,22 ). We now demonstrate that the MT1- MMP/PTK7 axis is essential for the polarized cell motility through the regulation of cell protrusions, including lamellipodia and invadopodia. "
    [Show abstract] [Hide abstract] ABSTRACT: It is well established that widely expressed PTK7 is essential for vertebrate tissue morphogenesis. In cancer, the functionality of PTK7 is selectively regulated by membrane type-1 matrix metalloproteinase (MT1-MMP), ADAMs (a disintegrin domain and metalloproteinases), and γ-secretase proteolysis. Here, we established that the full-length membrane PTK7, its Chuzhoi mutant with the two functional MT1-MMP cleavage sites, and its L622D mutant with the single inactivated MT1-MMP cleavage site differentially regulate cell motility in a two-dimensional versus three-dimensional environment. We also demonstrated that in polarized cancer cells, the levels of PTK7 expression and proteolysis were directly linked to the structure and kinetics of cell protrusions, including lamellipodia and invadopodia. In the functionally relevant and widely accepted animal models of metastasis, mouse and chick embryo models, both the overexpression and knock-out of PTK7 in HT1080 cells abrogated metastatic dissemination. Our analysis of human tissue specimens confirmed intensive proteolysis of PTK7 in colorectal cancer tumors, but not in matching normal tissue. Our results provide convincing evidence that both PTK7 expression and proteolysis, rather than the level of the cellular full-length PTK7 alone, contribute to efficient directional cell motility and metastasis in cancer.
    Full-text · Article · Jul 2014
    • "Human fibrosarcoma HT1080 cells (HT1080 cells) were from ATCC (Manassas, VA, USA). HT1080 cells transfected with the full-length 1–1070 PTK7 containing the C-terminal FLAG tag (PTK7 cells), the N-terminal 1–694 fragment with the C-terminal FLAG tag (sPTK7 cells) and the C-terminal 726–1070 fragment with the V5-HIS tag (cPTK7/726-1070 cells) were described earl- ier [11,21,23]. Cells in which the transcription of the MT1-MMP gene was silenced using the shRNA construct (shMT1 cells) were characterized earlier [11,45]. "
    [Show abstract] [Hide abstract] ABSTRACT: The full-length membrane protein tyrosine kinase 7 (PTK7) pseudokinase, an important component of the planar cell polarity and the Wnt canonical and non-canonical pathways, is a subject of step-wise proteolysis in cells and tissues. The proteolysis of PTK7 involves membrane type-matrix metalloproteinase (MT1-MMP), members of the Disintegrin Domain and Metalloproteinase (ADAM) family, and gamma-secretase. This multi-step proteolysis results in the generation of the digest fragments of PTK7. These fragments may be either liberated into the extracellular milieu or retained on the plasma membrane or released into the cytoplasm and then transported into the nucleus. We employed the genome-wide transcriptional and kinome array analyses to determine the role of the full-length membrane PTK7 and its proteolytic fragments in the downstream regulatory mechanisms, with an emphasis on the cell migration-related genes and proteins. Using fibrosarcoma HT1080 cells stably expressing PTK7 and its mutant and truncated species, the structure of which corresponded to the major PTK7 digest fragments, we demonstrated that the full-length membrane 1-1070 PTK7, the N-terminal 1-694 soluble ectodomain fragment, and the C-terminal 622-1070 and 726-1070 fragments differentially regulate multiple genes and signaling pathways in our highly invasive cancer cell model. Immunoblotting of the selected proteins were used to validate the results of our high throughput assays. Our results suggest that PTK7 levels need to be tightly controlled to enable migration and that the anti-migratory effect of the full-length membrane PTK7 is linked to the down-regulation of multiple migration-related genes and to the activation of the Akt and c-Jun pathway. In turn, the C-terminal fragments of PTK7 act predominantly via the RAS-ERK and CREB/ATF1 pathway and through the up-regulation of cadherin-11. In general, our data correlate well with the distinct functionality of the full-length receptor tyrosine kinases and their respective intracellular domain (ICD) proteolytic fragments.
    Full-text · Article · Mar 2014
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