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

The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP⁶²¹↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.     

Protein-tyrosine Pseudokinase 7 (PTK7) Directs Cancer Cell Motility and Metastasis

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.     

Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion.     

Potential relation of aberrant proteolysis of human protein tyrosine kinase 7 (PTK7) chuzhoi by membrane type 1 matrix metalloproteinase (MT1-MMP) to congenital defects

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.     

Fluid shear stress and sphingosine 1-phosphate activate calpain to promote membrane type 1 matrix metalloproteinase (MT1-MMP) membrane translocation and endothelial invasion into three-dimensional collagen matrices

The vascular endothelium continually senses and responds to biochemical and mechanical stimuli to appropriately initiate angiogenesis. We have shown previously that fluid wall shear stress (WSS) and sphingosine 1-phosphate (S1P) cooperatively initiate the invasion of human umbilical vein endothelial cells into collagen matrices (Kang, H., Bayless, K. J., and Kaunas, R. (2008) Am. J. Physiol. Heart Circ. Physiol. 295, H2087-2097). Here, we investigated the role of calpains in the regulation of endothelial cell invasion in response to WSS and S1P. Calpain inhibition significantly decreased S1P- and WSS-induced invasion. Short hairpin RNA-mediated gene silencing demonstrated that calpain 1 and 2 were required for WSS and S1P-induced invasion. Also, S1P synergized with WSS to induce invasion and to activate calpains and promote calpain membrane localization. Calpain inhibition results in a cell morphology consistent with reduced matrix proteolysis. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown by others to regulate endothelial cell invasion, prompting us to test whether calpain acted upstream of MT1-MMP. S1P and WSS synergistically activated MT1-MMP and induced cell membrane localization of MT1-MMP in a calpain-dependent manner. Calpain activation, MT1-MMP activation and MT1-MMP membrane localization were all maximal with 5.3 dynes/cm(2) WSS and S1P treatment, which correlated with maximal invasion responses. Our data show for the first time that 5.3 dynes/cm(2) WSS in the presence of S1P combine to activate calpains, which direct MT1-MMP membrane localization to initiate endothelial sprouting into three-dimensional collagen matrices.     

MT1-MMP: a potent modifier of pericellular microenvironment

Cells are regulated by many different means, and there is more and more evidence emerging that changes in the microenvironment greatly affect cell function. MT1-MMP is a type I transmembrane proteinase which participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules. The enzyme is cellular collagenase essential for skeletal development, cancer invasion, growth, and angiogenesis. MT1-MMP promotes cell invasion and motility by pericellular ECM degradation, shedding of CD44 and syndecan1, and by activating ERK. Thus MT1-MMP is one of the factors that influence the cellular microenvironment and thereby affect cell-signaling pathways and eventually alters cellular behavior. As a proteinase, MT1-MMP is regulated by inhibitors, but it also requires formation of a homo-oligomer complex, localization to migration front of the cells, and internalization to become a "functionally active" cell function modifier. Developing new means to inhibit "functional activity" of MT1-MMP may be a new direction to establish treatments for the diseases that MT1-MMP mediates such as cancer and rheumatoid arthritis.     

Phosphorylation of membrane type 1-matrix metalloproteinase (MT1-MMP) and its vesicle-associated membrane protein 7 (VAMP7)-dependent trafficking facilitate cell invasion and migration

In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.     

Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.     

Tissue inhibitor of metalloproteinases-2 binding to membrane-type 1 matrix metalloproteinase induces MAPK activation and cell growth by a non-proteolytic mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.     

Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis

Membrane type-1 matrix metalloproteinase (MT1-MMP) supports tumor cell invasion through extracellular matrix barriers containing fibrin, collagen, fibronectin, and other proteins. Here, we show that simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, and TSPAN12) markedly decreases MT1-MMP proteolytic functions in cancer cells. Affected functions include fibronectin proteolysis, invasion and growth in three-dimensional fibrin and collagen gels, and MMP-2 activation. Tetraspanin proteins (CD9, CD81, and TSPAN2) selectively coimmunoprecipitate and colocalize with MT1-MMP. Although tetraspanins do not affect the initial biosynthesis of MT1-MMP, they do protect the newly synthesized protein from lysosomal degradation and support its delivery to the cell surface. Interfering with MT1-MMP-tetraspanin collaboration may be a useful therapeutic approach to limit cancer cell invasion and metastasis.     

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

Cellular membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves C3b, an essential component of the complement system

Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream.     

Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase. Expression of the latent zymogen in Pichia pastoris,autolytic activation,and the peptide sequence of the cleavage forms

An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112). The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.     

SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) mediate trafficking of membrane type 1–matrix metalloproteinase (MT1-MMP) during invadopodium formation and tumor cell invasion

Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1–matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide–sensitive factor–activating protein receptor (SNARE)–mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.     

Membrane type 1 matrix metalloproteinase (MT1-MMP) ubiquitination at Lys581 increases cellular invasion through type I collagen

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP14) is a zinc-dependent type I transmembrane metalloproteinase playing pivotal roles in the regulation of pericellular proteolysis and cellular migration. Elevated expression levels of MT1-MMP have been demonstrated to correlate with a poor prognosis in cancer. MT1-MMP has a short intracellular domain (ICD) that has been shown to play important roles in cellular migration and invasion, although these ICD-mediated mechanisms remain poorly understood. In this study, we report that MT1-MMP is mono-ubiquitinated at its unique lysine residue (Lys(581)) within the ICD. Our data suggest that this post-translational modification is involved in MT1-MMP trafficking as well as in modulating cellular invasion through type I collagen matrices. By using an MT1-MMP Y573A mutant or the Src family inhibitor PP2, we observed that the previously described Src-dependent MT1-MMP phosphorylation is a prerequisite for ubiquitination. Taken together, these findings show for the first time an additional post-translational modification of MT1-MMP that regulates its trafficking and cellular invasion, which further emphasizes the key role of the MT1-MMP ICD.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.