Project description:Full-length membrane PTK7 and its N-terminal and C-terminal proteolytic fragments induced differential transcriptional profiles in HT1080 cells. Pseudokinase PTK7 is an essential regulator of planar cell polarity (PCP) and directional cell motility in the course of vertebrate development and embryogenesis, and regulates both the non-canonical Wnt/PCP and canonical Wnt pathways. In contrast to its well-appreciated, crucial role in embryo development, the functional importance of the intact full-length PTK7 in malignancy is still a matter of debate. In humans, the full-length membrane PTK7 consists of seven extracellular immunoglobulin-like (Ig) domains, a transmembrane region, a juxtamembrane region and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain. Because pericellular proteolysis plays a primary role in cell migration, especially in the directional locomotion of tumor cells, it is likely that proteolysis and PCP converge to promote efficient directed cancer cell migration. In agreement, the functionality of PTK7 is directly regulated by proteolysis. Ubiquitous membrane type-1 matrix metalloproteinase (MT1-MMP), arguably the primary enzyme in pericellular proteolysis and cancer cell migration, cleaves the PKP621↓LI sequence in the seventh Ig-like domain of membrane PTK7 and this cleavage results in the liberation of the N-terminal soluble PTK7 fragment (sPTK7). MT1-MMP proteolysis is followed by the cleavage of the C-terminal residual portion of PTK7 by ADAMs, including ADAM17. The ectodomain shedding is a prerequisite for the intramembrane cleavage of PTK7 by γ-secretase. This cleavage releases the C-terminal cytoplasmic tail fragment of PTK7, which is then either degraded by the proteasome or transported to the nucleus.The limited pre-existing data suggest that the full-length membrane PTK7 and its proteolytic products cause an opposing 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, in agreement, reduced migration efficiency of fibrosarcoma HT1080 cells. MT1-MMP proteolysis of PTK7 reversed the inhibitory effect of the full-length membrane PTK7, resulted in the accumulation of the stable N-terminal sPTK7 fragment in the extracellular milieu and promoted cell invasion of HT1080 cells. Expression of the Chz PTK7 mutant that exhibited an additional PEK↓LK503 MT1-MMP cleavage site in the junction region between the fifth and the sixth Ig-like domains stimulated cell migration even further. These effects suggest the existence of the intriguing and specific downstream mechanisms by which the intact PTK7, its digest fragments and the homo- and heterodimeric complexes between the PTK7 membrane, soluble and intracellular portions control cell function. These mechanisms, however, have not been precisely investigated in the earlier works by us and others. Understanding of these mechanisms will shed additional light on the role that PTK7 alone as well as in its combination with MT1-MMP, ADAMs and γ-secretase plays in cancer cell migration.

Project description:Full-length membrane PTK7 and its N-terminal and C-terminal proteolytic fragments induced differential transcriptional profiles in HT1080 cells. Pseudokinase PTK7 is an essential regulator of planar cell polarity (PCP) and directional cell motility in the course of vertebrate development and embryogenesis, and regulates both the non-canonical Wnt/PCP and canonical Wnt pathways. In contrast to its well-appreciated, crucial role in embryo development, the functional importance of the intact full-length PTK7 in malignancy is still a matter of debate. In humans, the full-length membrane PTK7 consists of seven extracellular immunoglobulin-like (Ig) domains, a transmembrane region, a juxtamembrane region and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain. Because pericellular proteolysis plays a primary role in cell migration, especially in the directional locomotion of tumor cells, it is likely that proteolysis and PCP converge to promote efficient directed cancer cell migration. In agreement, the functionality of PTK7 is directly regulated by proteolysis. Ubiquitous membrane type-1 matrix metalloproteinase (MT1-MMP), arguably the primary enzyme in pericellular proteolysis and cancer cell migration, cleaves the PKP621M-bM-^FM-^SLI sequence in the seventh Ig-like domain of membrane PTK7 and this cleavage results in the liberation of the N-terminal soluble PTK7 fragment (sPTK7). MT1-MMP proteolysis is followed by the cleavage of the C-terminal residual portion of PTK7 by ADAMs, including ADAM17. The ectodomain shedding is a prerequisite for the intramembrane cleavage of PTK7 by M-NM-3-secretase. This cleavage releases the C-terminal cytoplasmic tail fragment of PTK7, which is then either degraded by the proteasome or transported to the nucleus.The limited pre-existing data suggest that the full-length membrane PTK7 and its proteolytic products cause an opposing 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, in agreement, reduced migration efficiency of fibrosarcoma HT1080 cells. MT1-MMP proteolysis of PTK7 reversed the inhibitory effect of the full-length membrane PTK7, resulted in the accumulation of the stable N-terminal sPTK7 fragment in the extracellular milieu and promoted cell invasion of HT1080 cells. Expression of the Chz PTK7 mutant that exhibited an additional PEKM-bM-^FM-^SLK503 MT1-MMP cleavage site in the junction region between the fifth and the sixth Ig-like domains stimulated cell migration even further. These effects suggest the existence of the intriguing and specific downstream mechanisms by which the intact PTK7, its digest fragments and the homo- and heterodimeric complexes between the PTK7 membrane, soluble and intracellular portions control cell function. These mechanisms, however, have not been precisely investigated in the earlier works by us and others. Understanding of these mechanisms will shed additional light on the role that PTK7 alone as well as in its combination with MT1-MMP, ADAMs and M-NM-3-secretase plays in cancer cell migration. To evaluate in detail the effects of PTK7 and its proteolytic fragments on genome-wide transcriptional regulation, we specifically employed fibrosarcoma HT1080 cells. These highly invasive cells express low endogenous levels of PTK7 but high levels of active MT1-MMP and ADAMs. Because of these parameters, we could manipulate this preudokinase functionality using HT1080 cells transfected with the recombinant PTK7 constructs. The cells we employed included the PTK7 knock-out cells (shPTK7 cells), cells with the enforced overexpression of the original membrane PTK7 (PTK7 cells) and its Chuzhoi (Chz) mutant that exhibited an additional PEKM-bM-^FM-^SLK503 MT1-MMP cleavage site (Chz cells), and the cells, which overexpresssed multiple deletion species of PTK7. These species represented the soluble (sPTK7), membrane and cytoplasmic C-terminal digest fragments that resulted because of PTK7 cleavage by MT1-MMP (cPTK7/622-1070) and ADAM/gammaM-bM-^@M-^Ssecretase (cPTK7/726-1070). Expression of these constructs allowed us to specifically determine the downstream effect of PTK7 proteolysis on gene expression. HT1080 cells were stably transfected with the PTK7 constructs. Cells (1 x 10^4/ml) were plated in DMEM- 10% FBS in a 100-mm dish and grown for 72 h to produce a subconfluent culture. Total cellular RNA was extracted using a Direct-zol RNA MiniPrep kit (Zymo Research). Biotin-labeled cRNA samples were prepared using the Illumina RNA Amplification Kit (Ambion). The labeled cRNA (750 ng) was hybridized for 18 h at 58M-BM-0C to the HumanHT-12 v4 Expression BeadChip (Illumina). BeadChips were then developed using fluorolink streptavidin-Cy3 (GE Healthcare). Array chips were scanned using an Illumina BeadArray Reader. The initial data extraction and normalization were performed using the BeadArray Reader and GeneSpring GX software (Agilent).

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The Wnt co-receptor PTK7/Otk in Neurogenesis and Patterning

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