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

Abstract

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.About three of every 100 babies born in the United States have congenital abnormalities. Congenital abnormalities are ranked 34th of the top 50 causes of total death in the United States. Debilitating cardiovascular and nervous system defects jointly represent 40% of birth defects. The death toll of congenital abnormalities (0.58%) roughly equals that of stomach, skin, or oral cancers. Mechanistic research into birth defects is required to more efficiently contribute to prevention and correction of these disorders.The canonical, ␤-catenin-dependent and non-canonical, ␤-catenin-independent Wnt signaling pathways are conserved in eukaryotes and are critical for the diverse events in embryonic development and disease. The non-canonical Wnt/planar cell polarity (PCP) 2 signaling controls cell organization within the tissue plane (1, 2). The Wnt/PCP signaling affects the actin cytoskeleton via RhoA GTPase activation through dishevelled associated activator of morphogenesis 1 (DAAM1) and dishevelled activator of morphogenesis 2 (DAAM2). The first PCP signaling events occur at a gastrulation stage of embryogenesis. These events regulate the polarized directed cell movement to accomplish convergent extension for the anterior-posterior body axis elongation, neural tube closure, and craniofacial morphogenesis (3-6). Convergent extension failure results in a shortened anterior-posterior body axis and widened lateral axis (convergent extension phenotype), a defective neural system, and craniofacial abnormalities.Ubiquitously expressed PTK7 (also called colon carcinoma kinase-4 (CCK-4)) is a Wnt coreceptor and a regulator of PCP in vertebrates (7-13). Membrane PTK7 includes seven extracellular Ig domains, a transmembrane region, and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain (8,9,11,14). PTK7 is evolutionary conserved in humans and mice and also chicken klingon (KLG...

Insights into Ectodomain Shedding and Processing of Protein-tyrosine Pseudokinase 7 (PTK7)

“…Thus, the Chz PTK7 mutant exhibited an additional MMP cleavage site in the ectodomain. As compared with the wild-type and especially L622D mutant PTK7, the Chz mutant was efficiently shed from the cell surface and promoted cell invasion (43). We determined that the PTK7 ectodomain was a major cleavage target of invasion-promoting MT1-MMP at the cell surface.…”

“…We suspect that this effect is a result of the repressed ECM degradation. On the other hand, overexpression of the PTK7-FLAG that reduced cytoskeleton contractility (43,44) inhibited cell invasion also by 50%, although the MT1-MMP activity was not affected. Accordingly, we now suspect that this effect we observed in the HT1080 PTK7-FLAG cells is linked to the PTK7 effect on the cytoskeleton rather than related to ECM degradation.…”

“…Cloning and Mutagenesis-HT1080 cells transfected with the full-length MT1-MMP (MT1-MMP cells), the full-length PTK7-V5-HIS and PTK7-FLAG constructs (PTK7-V5 and PTK7-FLAG cells, respectively), and the L622D-FLAG PTK7 mutant with the inactivated MT1-MMP cleavage site (L622D-FLAG cells) were characterized earlier (43,44). The shRNA constructs for the MT1-MMP silencing and the required scrambled shRNA controls were obtained and characterized earlier (33,45,46).…”

“…Thus, the PTK7 ectodomain was efficiently shed in a variety of cell lines, in which MT1-MMP was overexpressed, and also in human fibrosarcoma HT1080 cells with high endogenous levels of MT1-MMP (43,44). MT1-MMP cleaved the exposed PKP 621 2LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generated, as a result, an N-terminal, soluble, 65-kDa ectodomain fragment (N-PTK7-65) and the C-terminal membrane-attached, 50-kDa fragment (C-PTK7-50) that included the pseudokinase domain.…”

“…MT1-MMP cleaved the exposed PKP 621 2LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generated, as a result, an N-terminal, soluble, 65-kDa ectodomain fragment (N-PTK7-65) and the C-terminal membrane-attached, 50-kDa fragment (C-PTK7-50) that included the pseudokinase domain. MT1-MMP proteolysis of PTK7 reversed the anti-invasive function of the full-length PTK7 (43,44).…”