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

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 2LI 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.Secreted Wnt glycoproteins regulate ␤-catenin-dependent (canonical) and ␤-catenin-independent (non-canonical) signaling pathways (1-7). One intriguing and well conserved function of the non-canonical pathway is to control PCP 2 and directional 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 collective 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-posterior 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 abnormalities. 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-canonical 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 aberrant regulation in disease is required to control tumor progression and metastasis in a clinically advantageous man...

Potential Relation of Aberrant Proteolysis of Human Protein Tyrosine Kinase 7 (PTK7) chuzhoi by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) to Congenital Defects

“…Consistent with our earlier results (29), the ectopic expression of PTK and especially the uncleavable L622D PTK7 mutant suppressed cell migration (Fig. 4).…”

“…Cell surface levels of PTK7 appear to correlate inversely with tumor aggressiveness and metastatic potential (29). Because of these earlier data and because actin cytoskeleton dynamics are also associated with cell migration (44,45), we next analyzed the invasive capacity of HT1080, HT-PTK7, HT-L622D, HT-sPTK7, HT-chz, and HT-siMT1/chz cells.…”

“…Gelatin Substrate Zymography of MMP-2 and Western Blotting of RhoA, PTK7, and MT1-MMP-Basic protocols for these techniques have been described previously (29). The purified MMP-2 proenzyme (100 ng/ml) was added to MCF7 cells 12-14 h prior to the gelatin zymography analysis (35).…”

“…The C-terminally tagged (with a V5 tag) wild type and catalytically inert (E240A) mutant MT1-MMP constructs (29,32) were used to generate the MCF7-MT1 and MCF7-E240A cells, respectively. HT1080 cells with the ectopic expression of the FLAG-tagged PTK7 constructs, including the full-length wild-type PTK7 (HT-PTK7), the uncleavable PTK7 L622D mutant (HT-L622D), and the N-terminal-soluble, 1-700 ectodomain form of PTK7 (HT-sPTK7), were obtained and characterized earlier (29). The PTK7-FLAG template was used in the PCR reactions with the following primers: forward, 5Ј-CGTGTCCAAGTGCTGGCTAA-CCCAGAAAAGCTCAAGTTC-3Ј; and reverse, 5Ј-GAACTT-GAGCTTTTCTGGGTTAGCCAGCACTTGGACACG-3Ј to generate the chz mutant sequence (the mutant sequence is underlined).…”

“…It becomes now possible to suggest that the N-terminal, soluble (70-kDa) 1-621 chz fragment readily degrades in vivo. This degradation of soluble chz leads to the aberrant membrane/soluble PTK ratio and to the significantly modified migratory and signaling cell characteristics (17,29).…”