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

Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2010; 285(46):35740-9. DOI: 10.1074/jbc.M110.165159
Source: PubMed


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.

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Available from: Vladislav Golubkov, Feb 18, 2014
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    • "The limited pre-existing data suggest that the full-length membrane PTK7 and its proteolytic products cause a contrasting effect on the efficiency of cell migration. Thus, the continuing presence of the full-length membrane PTK7 on the plasma membrane down-regulated the myosin light chain (MLC) phosphorylation (a downstream event of the Wnt/PCP pathway) and also reduced migration efficiency of fibrosarcoma HT1080 cells [21]. MT1-MMP proteolysis of PTK7 reversed the inhibitory effect of the full-length membrane PTK7, and resulted in the accumulation of the Stable N-terminal sPTK7 fragment in the extracellular milieu and promoted cell invasion of HT1080 cells [11,21,23]. "
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    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.
    Cell Communication and Signaling 03/2014; 12(1):15. DOI:10.1186/1478-811X-12-15 · 3.38 Impact Factor
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    • "HT1080 cells stably transfected with the full-length MT1–MMP (HT1080-MT1 cells), the MT1–MMP-silencing small hairpin RNA construct (HT1080-shMT1 cells) and the full-length PTK7-FLAG construct (HT1080-PTK7 cells) were characterized earlier.20, 21, 22 "
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    ABSTRACT: Matrix metalloproteinases (MMPs) and, especially membrane type 1 (MT1)-MMP/MMP-14, are promising drug targets in malignancies. In contrast with multiple small-molecule and protein pan-inhibitors of MT1-MMP cleavage activity, the murine 9E8 monoclonal antibody targets the MMP-2-activating function of cellular MT1-MMP alone, rather than the general proteolytic activity and the pro-migratory function of MT1-MMP. Furthermore, the antibody does not interact in any detectable manner with other members of the membrane type (MT)-MMP family. The mechanism of this selectivity remained unknown. Using mutagenesis, binding and activity assays, and modeling in silico, we have demonstrated that the 9E8 antibody recognizes the MT-loop structure, an eight residue insertion that is specific for MT-MMPs and that is distant from the MT1-MMP active site. The binding of the 9E8 antibody to the MT-loop, however, prevents tissue inhibitor of metalloproteinases-2 (TIMP-2) association with MT1-MMP. As a result, the 9E8 antibody incapacitates the TIMP-2-dependent MMP-2-activating function alone rather than the general enzymatic activity of human MT1-MMP. The specific function of the 9E8 antibody we determined directly supports an essential, albeit paradoxical, role of the protein inhibitor (TIMP-2) in MMP-2 activation via a unique membrane-tethered mechanism. In this mechanism, the formation of a tri-molecular MT1-MMPTIMP-2MMP-2 complex is required for both the capture of the soluble MMP-2 proenzyme by cells and then its well-controlled conversion into the mature MMP-2 enzyme. In sum, understanding of the structural requirements for the 9E8 antibody specificity may pave the way for the focused design of the inhibitory antibodies against other individual MMPs.
    Oncogenesis 12/2013; 2(12):e80. DOI:10.1038/oncsis.2013.44 · 3.95 Impact Factor
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    • "PTK7 has been shown to play critical roles in developmental processes, most notably the regulation of gastrulation and neural tube closure [20], [21]. Both of these events entail convergent extension, which involves the regulation of planar cell polarity [22], [23], in which PTK7 is a crucial regulator [21], [24], [25]. In addition, PTK7 is also involved in other developmental events such as axon guidance, neural crest migration and cardiac morphogenesis [26], [27], [28], [29], [30]. "
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    ABSTRACT: Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development, but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak, we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24-48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs, and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population, which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting, dissociation and re-plating reestablishes an epithelial phenotype.
    PLoS ONE 11/2012; 7(11):e50432. DOI:10.1371/journal.pone.0050432 · 3.23 Impact Factor
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