Identification and Characterization of Lutheran Blood Group Glycoprotein as a New Substrate of Membrane-type 1 Matrix Metalloproteinase 1 (MT1-MMP): A SYSTEMIC WHOLE CELL ANALYSIS OF MT1-MMP-ASSOCIATING PROTEINS IN A431 CELLS*

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.Cells in tissues are surrounded by an extracellular cellular matrix that interacts with cells to regulate their activity (1, 2). Matrix metalloproteinases (MMPs)³ are endopeptidases responsible for extracellular matrix degradation and thereby regulate turnover of the extracellular matrix. However, recent studies have demonstrated that substrates of MMPs are expanded to a variety of pericellular proteins.MT1-MMP/MMP14 is an integral membrane proteinase that cleaves multiple proteins in the pericellular milieu and thereby regulates various cell functions. Substrates of MT1-MMP identified to date include extracellular matrix proteins (type I collagen, fibronectin, vitronectin, laminin-1 and -5, and others), cell adhesion molecules (CD44, syndecan-1, and αv integrin), cytokines (SDF-1 and transforming growth factor-β and others), and latent forms of pro-MMPs (pro-MMP-2 and pro-MMP13) (3–5). Processing of these proteins by MT1-MMP alters their activities and thereby regulates a variety of cellular functions, such as motility, invasion, growth, differentiation, and apoptosis. Consistent with these functions, forced expression of MT1-MMP in tumor cells enhances behavior consistent with increased malignancy, such as rapid tumor growth, invasion, and metastasis (6). However, MT1-MMP is normally expressed in various types of cell and mice deficient in MT1-MMP expression (MT1^−/−) display pleiotropic defects (7–10). However, we as yet have only limited knowledge of the physiological substrates of MT1-MMP that could explain such pleiotropic effects.Proteases interact with their substrates at least transiently, but in some cases such interaction is more stable. For instance, type I collagen binds MT1-MMP via a hemopexin-like domain and is cleaved (11, 12). Cleavage of collagen by MT1-MMP regulates cell growth and invasion in a collagen-rich environment (13). CD44, a hyaluronic acid receptor, also binds to the hemopexin of MT1-MMP and is cleaved (14). Expression of CD44 and MT1-MMP in tumor cells promotes cell migration, accompanied by the shedding of CD44 by MT1-MMP (14, 15). pro-MMP-2, which is cleaved by MT1-MMP for activation, forms a tri-molecular complex with MT1-MMP and TIMP-2 (3, 16). Therefore, screening of proteins that associate with MT1-MMP may provide a systematic method to identify potential substrates of MT1-MMP in cells. In addition, these proteins may also be regulatory proteins of MT1-MMP.To identify proteins associating with MT1-MMP in different types of tumor cells, we first studied conditions for cell lysis using malignant melanoma A375 cells and following purification method of the proteins as reported recently (17). Proteins purified in this manner were analyzed by high-throughput proteomic analysis (18–21). Interestingly, approximately one-half of the membrane proteins identified in our previous study could be cleaved by MT1-MMP at least in vitro. Here, we applied this approach to human carcinoma cells (A431) that originate from epidermoid cells and further validated the systemic whole cell analysis method. To evaluate whether the MT1-MMP-associated membrane proteins so identified include physiological targets of MT1-MMP activity, we select one of them, Lutheran blood group glycoprotein (Lu), and evaluate its processing in A431 cells.     

Modulation of the Membrane Type 1 Matrix Metalloproteinase Cytoplasmic Tail Enhances Tumor Cell Invasion and Proliferation in Three-dimensional Collagen Matrices

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phos pho ryl a tion and that this modification may influence its enzymatic activity at the cell surface. In this study, phos pho ryl a ted MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phos pho ryl a ted by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr⁵⁶⁷ in these processes, mutants that mimic a state of either constitutive (T567E) or defective phos pho ryl a tion (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr⁵⁶⁷ exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phos pho ryl a tion at cytoplasmic tail residue Thr⁵⁶⁷ and that this modification plays a critical role in processes that are linked to tumor progression.Largely composed of a mixture of collagens, laminins, and vitronectin, the extracellular matrix (ECM)² serves as both a physical scaffold and a barrier against cell invasion. It has become increasingly evident that the structural condition of the ECM plays a unique role in regulating cell behavior. Proteolysis of integral components of the basement membrane disturbs the barrier provided by the ECM. Without physical restriction, cells invade the surrounding environment in an unregulated manner. The ability of matrix metalloproteinases (MMPs) to collectively degrade nearly all ECM constituents allows this class of enzymes to function in a diverse range of physiological processes (1, 2). Of the anchored MMPs, membrane type 1 matrix metalloproteinase (MT1-MMP) was the first to be discovered and has been most thoroughly characterized. Unlike soluble MMPs, MT1-MMP has a stretch of hydrophobic amino acids that traverse the cell membrane, followed by a short cytoplasmic tail composed of 20 amino acids (3). The advantage of cell surface localization is 2-fold. Surface restriction allows MT1-MMP to modify the immediate pericellular environment, overcoming physical constraints imposed by the ECM (2). Localization at the cell surface also places tethered MMPs in an optimal position to function at invadapodia, highly specialized areas of the cell membrane that form during focalized cell invasion (4). Although information regarding the role of the cytoplasmic tail is relatively limited (5, 6), this domain may function as a bridge to the intracellular machinery.MT1-MMP has an essential role in matrix remodeling during physiological processes (7, 8). Conversely, its enzymatic activity is key to acquiring a metastatic phenotype in a variety of tumor cells, including lung, colon, breast, and cervical carcinomas (2, 9–11). The ability to alter the physical structure of the pericellular environment, while triggering the activation and modification of several cell surface proteins, identifies a central role for MT1-MMP in influencing cellular behavior (12). In return, stringent cellular regulation of MT1-MMP enzymatic activity is necessary to prevent aberrant proteolysis.Increasing evidence suggests that the cytoplasmic tail of MT1-MMP may regulate its activity at the cell surface. It has been demonstrated that MT1-MMP is internalized from the cell surface and that this process requires the presence of the cytoplasmic domain (5, 6). Tail truncation restricts MT1-MMP to the cell surface, suggesting that this domain contains sequence(s) that either mediate internalization or are required for physical interaction with another protein that facilitates its internalization (5, 6). The mechanism regulating this process has yet to be determined. Interestingly, both invasion and migration are down-regulated in cells where MT1-MMP is restricted to the cell surface (5, 6). These data suggest a correlation between internalization and matrix turnover, where MT1-MMP activity is either abrogated or enhanced under appropriate stimuli.Reversible phosphorylation is widely recognized as a key post-translational modification that regulates protein function. The cytoplasmic domain of MT1-MMP has three potential phosphorylation sites: Thr⁵⁶⁷, Tyr⁵⁷³, and Ser⁵⁷⁷. Recent work by Nyalendo et al. (13) indicates that MT1-MMP is phosphorylated at tyrosine residue Tyr⁵⁷³, and that this modification influences cell migration. Several surface proteins are regulated by phosphorylation at multiple residues. In the MT1-MMP cytoplasmic tail, Thr⁵⁶⁷ has homology with the consensus sequence for both protein kinase C (TXR) and ERK1/2 (XTP) (14), suggesting the possibility that active MT1-MMP might also be regulated through phosphorylation of this cytoplasmic tail residue. In the present study, we report that MT1-MMP bears a threonine phosphorylation site in its cytoplasmic tail and that this modification plays an important role in regulating several aspects of carcinoma cell behavior, including invasion and three-dimensional growth.     

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg⁹⁸ and Arg¹⁰¹ residues, whereas plasmin only cleaved the propeptide downstream of Arg¹⁰¹. Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn¹⁰⁹-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.Matrix metalloprotease (MMP)³-2 is a member of the zinc-dependent endopeptidase family, which comprises 24 enzymes (1). MMP-2 plays a key role in many biological and pathological processes, including organ growth, endometrial cycling, wound healing, bone remodeling, tumor invasion, and metastasis (2). This enzyme functions through proteolysis of non-structural extracellular molecules and components of the basement membrane, including type IV collagen, fibronectin, elastin, laminin, aggrecan, and fibrillin (3).Like most MMPs, MMP-2 is synthesized as a zymogen that is activated by conformational change (4) or proteolysis within the propeptide, which may involve membrane type MMPs (MT-MMPs) (5–9). The most studied activation mechanism for pro-MMP-2 is cleavage of the propeptide by MT1-MMP, which requires cooperative activity between MT1-MMP and tissue inhibitor of metalloprotease (TIMP)-2 (5, 10–12). Serine proteases, such as thrombin, factor Xa, activated protein C, and plasmin as well as the cysteine protease legumain are all known activators of pro-MMP-2 (13–17).In addition to its role in coagulation, thrombin is involved in multiple cellular processes, including mitogenesis of fibroblasts (18), lymphocytes (19), mesenchymal cells (20), and smooth muscle cells (SMCs) (21, 22). Factor Xa acts as a potent mitogen for endothelial cells (23), fibroblasts (24), and vascular SMCs (25, 26). Both proteases can also elicit endothelial cell and SMC migration through pro-MMP-2 activation and subsequent extracellular matrix degradation (13, 27, 28). However, despite studies suggesting that MT1-MMP is involved in thrombin-mediated activation of pro-MMP-2, a detailed mechanism for MMP-2 activation has yet to be elucidated (15, 27).In this study, we investigated the roles of factor Xa and thrombin in MMP-2 regulation. Data are presented to demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 by cleavage of specific sites within the propeptide. Furthermore, factor Xa-processed MMP-2 showed enzymatic activity that was enhanced following intermolecular autoproteolytic cleavage. Thrombin also activated pro-MMP-2 through the same cleavage reaction. Interestingly, factor Xa and thrombin were also found to be involved in MMP-2 degradation. However, this activity was reduced greatly in thrombin-treated MMP-2 by the cell surface, which resulted in an increase in processed MMP-2.     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.     

The Amino-terminal Domain of Desmoplakin Binds to Plakoglobin and Clusters Desmosomal Cadherin–Plakoglobin Complexes

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.Desmosomes are highly organized adhesive intercellular junctions that couple intermediate filaments to the cell surface at sites of cell–cell adhesion (Farquhar and Palade, 1963; Staehelin, 1974; Schwarz et al., 1990; Garrod, 1993; Collins and Garrod, 1994; Cowin and Burke, 1996; Kowalczyk and Green, 1996). Desmosomes are prominent in tissues that experience mechanical stress, such as heart and epidermis, and the disruption of desmosomes or the intermediate filament system in these organs has devastating effects on tissue integrity (Steinert and Bale, 1993; Coulombe and Fuchs, 1994; Fuchs, 1994; McLean and Lane, 1995; Stanley, 1995; Bierkamp et al., 1996; Ruiz et al., 1996). Desmosomes are highly insoluble structures that can withstand harsh denaturing conditions (Skerrow and Matoltsy, 1974; Gorbsky and Steinberg, 1981; Jones et al., 1988; Schwarz et al., 1990). This property of desmosomes facilitated early identification of desmosomal components but has impaired subsequent biochemical analysis of the protein complexes that form between desmosomal components. Ultrastructurally, desmosomes contain a core region that includes the plasma membranes of adjacent cells and a cytoplasmic plaque that anchors intermediate filaments to the plasma membrane. The plaque can be further divided into an outer dense plaque subjacent to the plasma membrane and an inner dense plaque through which intermediate filaments appear to loop.Molecular genetic analysis has revealed that the desmosomal glycoproteins, the desmogleins and desmocollins, are members of the cadherin family of cell–cell adhesion molecules (for review see Buxton et al., 1993, 1994; Cowin and Mechanic, 1994; Kowalczyk et al., 1996). The classical cadherins, such as E-cadherin, mediate calcium-dependent, homophilic cell–cell adhesion (Nagafuchi et al., 1987). The mechanism by which the desmosomal cadherins mediate cell–cell adhesion remains elusive (Amagai et al., 1994; Chidgey et al., 1996; Kowalczyk et al., 1996), although heterophilic interactions have recently been detected between desmogleins and desmocollins (Chitaev and Troyanovsky, 1997). Both classes of the desmosomal cadherins associate with the cytoplasmic plaque protein plakoglobin (Kowalczyk et al., 1994; Mathur et al., 1994; Roh and Stanley, 1995b ; Troyanovsky et al., 1994), which is part of a growing family of proteins that share a repeated motif first identified in the Drosophila protein Armadillo (Peifer and Wieschaus, 1990). This multigene family also includes the desmosomal proteins band 6/plakophilin 1, plakophilin 2a and 2b, and p0071, which are now considered to comprise a subclass of the armadillo family of proteins (Hatzfeld et al., 1994; Heid et al., 1994; Schmidt et al., 1994; Hatzfeld and Nachtsheim, 1996; Mertens et al., 1996).The most abundant desmosomal plaque protein is desmoplakin, which is predicted to be a homodimer containing two globular end domains joined by a central α-helical coiled-coil rod domain (O''Keefe et al., 1989; Green et al., 1990; Virata et al., 1992). Previous studies have demonstrated that the carboxyl-terminal domain of desmoplakin interacts with intermediate filaments (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; Kouklis et al., 1994; Meng et al., 1997), and the amino-terminal domain of desmoplakin is required for desmoplakin localization to the desmosomal plaque (Stappenbeck et al., 1993). Direct evidence supporting a role for desmoplakin in intermediate filament attachment to desmosomes was provided recently when expression of an amino-terminal polypeptide of desmoplakin was found to displace endogenous desmoplakin from cell borders and disrupt intermediate filament attachment to the cell surface in A431 epithelial cell lines (Bornslaeger et al., 1996).The classical cadherins, such as E-cadherin, bind directly to both β-catenin and plakoglobin (Aberle et al., 1994; Jou et al., 1995; for review see Cowin and Burke, 1996). β-Catenin is also an armadillo family member (McCrea et al., 1991; Peifer et al., 1992), and both plakoglobin and β-catenin bind directly to α-catenin (Aberle et al., 1994, 1996; Jou et al., 1995; Sacco et al., 1995; Obama and Ozawa, 1997). α-Catenin is a vinculin homologue (Nagafuchi et al., 1991) and associates with both α-actinin and actin (Knudson et al., 1995; Rimm et al., 1995; Nieset et al., 1997). Through interactions with β- and α-catenin, E-cadherin is coupled indirectly to the actin cytoskeleton, and this linkage is required for the adhesive activity of E-cadherin (Ozawa et al., 1990; Shimoyama et al., 1992). In addition, E-cadherin association with plakoglobin appears to be required for assembly of desmosomes (Lewis et al., 1997), underscoring the importance of E-cadherin in the overall program of intercellular junction assembly. However, the hierarchy of molecular interactions that couple the desmosomal cadherins to the intermediate filament cytoskeleton is largely unknown, although the desmocollin cytoplasmic domain appears to play an important role in recruiting components of the desmosomal plaque (Troyanovsky et al., 1993, 1994). Since desmosomal cadherins form complexes with plakoglobin and because the amino-terminal domain of desmoplakin is required for desmoplakin localization at desmosomes, we hypothesized that the amino-terminal domain of desmoplakin interacts with the desmosomal cadherin– plakoglobin complex.In previous studies, we used L-cell fibroblasts to characterize plakoglobin interactions with the cytoplasmic domains of the desmosomal cadherins and found that the desmosomal cadherins regulate plakoglobin metabolic stability (Kowalczyk et al., 1994) but do not mediate homophilic adhesion (Kowalczyk et al., 1996). To test the ability of the desmoplakin amino-terminal domain to interact with the desmosomal cadherin–plakoglobin complex, we established a series of L-cell lines expressing the desmosomal cadherins in the presence or absence of a desmoplakin amino-terminal polypeptide (DP-NTP).¹ The results indicate that one important function of the desmoplakin amino-terminal domain is to cluster desmosomal cadherin–plakoglobin complexes. In addition, DP-NTP and plakoglobin were found to form complexes that could be co-immunoprecipitated from L-cell lysates. Using the yeast two hybrid system, DP-NTP was found to bind directly to plakoglobin but not Dsg1. These data suggest that plakoglobin couples the amino-terminal domain of desmoplakin to the desmosomal cadherins and that desmoplakin plays an important role in organizing the desmosomal cadherin–plakoglobin complex into discrete plasma membrane domains.     

Sec35p,a Novel Peripheral Membrane Protein,Is Required for ER to Golgi Vesicle Docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.Protein transport through the secretory pathway occurs via transport vesicles under the direction of a large set of protein components (Rothman, 1994). The process can be divided into three stages: (a) vesicle budding, (b) vesicle docking, and (c) membrane fusion, with distinct sets of proteins mediating each phase. The budding step involves recruitment of coat proteins to the membrane and culminates with the release of coated vesicles (Schekman and Orci, 1996). The docking reaction is likely to require a set of integral membrane proteins on the vesicle and target membranes, termed v-SNAREs¹ and t-SNAREs (vesicle- and target membrane-soluble N-ethylmaleimide–sensitive fusion protein [NSF] attachment protein [SNAP] receptors, respectively), that are thought to confer specificity through their pair-wise interactions (Söllner et al., 1993b ). Small GTP-binding proteins of the rab family also assist in the docking process (Ferro-Novick and Novick, 1993), but their precise function is not known. The fusion step ensues after docking and results in the delivery of the vesicular cargo to the next compartment in the secretory pathway.Vesicular transport from the ER to the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively characterized. Transport vesicle budding involves the assembly of the COPII coat, composed of the Sec13p/Sec31p (Pryer et al., 1993; Salama et al., 1993; Barlowe et al., 1994) and Sec23p/Sec24p heterodimers (Hicke and Schekman, 1989; Hicke et al., 1992), under the direction of an integral membrane protein, Sec12p (Nakano et al., 1988; Barlowe and Schekman, 1993), a small GTPase, Sar1p (Nakano and Muramatsu, 1989), and a multidomain protein, Sec16p (Espenshade et al., 1995; Shaywitz et al., 1997). Docking is thought to require a tethering event mediated by Uso1p (Cao et al., 1998), the yeast homologue of mammalian p115 (Barroso et al., 1995; Sapperstein et al., 1995), followed by or concurrent with the interaction of a set of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p (Newman and Ferro-Novick, 1987; Newman et al., 1990; Ossig et al., 1991; Shim et al., 1991; Søgaard et al., 1994) and perhaps Ykt6p (Søgaard et al., 1994; McNew et al., 1997), with the cognate t-SNARE on the Golgi, Sed5p (Hardwick and Pelham, 1992). For some time it was thought that fusion may be initiated by disassembly of the v/t-SNARE complex (Söllner et al., 1993a ) by yeast SNAP, Sec17p, (Griff et al., 1992) and NSF, Sec18p (Eakle et al., 1988; Wilson et al., 1989). However, this concept has been challenged by studies with a yeast system that reconstitutes homotypic vacuolar fusion, which suggests the action of Sec18p is before vesicle docking (Mayer et al., 1996; Mayer and Wickner, 1997). In addition, a prefusion role for NSF has been supported by the recent finding that liposomes bearing SNAREs alone can fuse in the absence of NSF (Weber et al., 1998).Several proteins involved in the regulation of yeast ER to Golgi v/t-SNARE complex assembly have been identified, including Ypt1p, Uso1p, and Sly1p. Ypt1p is a member of the rab family of small GTP-binding proteins that have been identified as important components of almost every stage in the secretory pathway (Ferro-Novick and Novick, 1993). Hydrolysis of GTP by rab-like proteins has been hypothesized to provide the regulatory switch that controls the fidelity of vesicular transport (Bourne, 1988). A second protein, Uso1p (Nakajima et al., 1991), appears to function in the same pathway as Ypt1p (Sapperstein et al., 1996), and both proteins have been demonstrated to be essential for SNARE complex assembly (Søgaard et al., 1994; Sapperstein et al., 1996; Lupashin and Waters, 1997). The third protein, Sly1p, is associated with the t-SNARE Sed5p (Søgaard et al., 1994). SLY1 is an essential gene in yeast (Dascher et al., 1991; Ossig et al., 1991), and Sly1p is required for ER to Golgi transport in vitro (Lupashin et al., 1996) and in vivo (Ossig et al., 1991). However, several lines of evidence, particularly from Sly1p homologues in other organisms, indicate that Sly1p may also function as a negative regulator of v/t-SNARE complex assembly, perhaps by preventing the association of the v- and t-SNAREs (Hosono et al., 1992; Pevsner et al., 1994; Schulze et al., 1994). A dominant allele of SLY1, termed SLY1-20, is capable of suppressing mutations in YPT1 and USO1, including complete deletions (Dascher et al., 1991; Sapperstein et al., 1996). Thus, in the presence of Sly1-20p, two components required for SNARE complex assembly are no longer essential. We have proposed a model (Sapperstein et al., 1996; Lupashin and Waters, 1997) in which Ypt1p and Uso1p function to relieve the inhibitory action of Sly1p on SNARE complex assembly. In this model Sly1-20p can be thought of as a noninhibitory form of SLY1 that renders Ypt1p and Uso1p superfluous.We believe that the ability of SLY1-20 to suppress defects in upstream docking regulators can be used to identify additional components involved in the regulation of vesicular docking. We have undertaken a genetic screen (to be presented elsewhere) to isolate novel components in this pathway which, when mutated, depend upon Sly1-20p for viability. In the course of this work, we discovered that two recently identified mutants, sec34 and sec35, can be suppressed by SLY1-20 and thus satisfy the criterion of our screen. These mutants were isolated in a novel screen to identify components involved in transport at any step between the ER and the trans-Golgi network (i.e., the Kex2p compartment) in yeast (Wuestehube et al., 1996). Both sec34 and sec35 accumulate the core-glycosylated form of secretory proteins at the nonpermissive temperature, indicating a block in ER to Golgi transport. Furthermore, electron microscopy indicated that both sec34 and sec35 accumulate numerous vesicles upon shift to the restrictive temperature (Wuestehube et al., 1996), a hallmark of genes whose protein products are involved in the docking or fusion phase of transport (Kaiser and Schekman, 1990). In this report we describe the cloning of SEC35 and analysis of its genetic interactions with other secretory genes. Strong genetic interaction between SEC35 and SLY1, YPT1, and USO1 suggests that Sec35p may function in vesicle docking. To test this possibility, we devised an in vitro transport assay that depends on the addition of purified Sec35p and Uso1p. Vesicles synthesized in the absence of functional Sec35p do not fuse with the Golgi compartment and remain as freely diffusible intermediates. Upon addition of Sec35p and Uso1p, vesicles dock to the Golgi and proceed to membrane fusion. Requirements for Sec35p at the vesicle docking step correlates our genetic experiments with the biochemically distinguishable steps of vesicle docking and membrane fusion.     

Secreted Versus Membrane-anchored Collagenases: RELATIVE ROLES IN FIBROBLAST-DEPENDENT COLLAGENOLYSIS AND INVASION*

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13^−/−, Mmp8^−/−, Mmp2^−/−, Mmp9^−/−, Mmp14^−/− and Mmp16^−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14^−/− or Mmp16^−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14^−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.In the postnatal state, fibroblasts are normally embedded in a self-generated three-dimensional connective tissue matrix composed largely of type I collagen, the major extracellular protein found in mammals (1–3). Type I collagen not only acts as a structural scaffolding for the associated mesenchymal cell populations but also regulates gene expression and cell function through its interactions with collagen binding integrins and discoidin receptors (2, 4). Consistent with the central role that type I collagen plays in defining the structure and function of the extracellular matrix, the triple-helical molecule is resistant to almost all forms of proteolytic attack and can display a decades-long half-life in vivo (4–6). Nonetheless, fibroblasts actively remodel type I collagen during wound healing, inflammation, or neoplastic states (2, 7–13).To date type I collagenolytic activity is largely confined to a small subset of fewer than 10 proteases belonging to either the cysteine proteinase or matrix metalloproteinase (MMP)² gene families (4, 14–18). As all collagenases are synthesized as inactive zymogens, complex proteolytic cascades involving serine, cysteine, metallo, and aspartyl proteinases have also been linked to collagen turnover by virtue of their ability to mediate the processing of the pro-collagenases to their active forms (13, 15, 19). After activation, each collagenase can then cleave native collagen within its triple-helical domain, thus precipitating the unwinding or “melting” of the resulting collagen fragments at physiologic temperatures (4, 15). In turn, the denatured products (termed gelatin) are susceptible to further proteolysis by a broader class of “gelatinases” (4, 15). Collagen fragments are then either internalized after binding to specific receptors on the cell surface or degraded to smaller peptides with potent biological activity (20–24).Previous studies by our group as well as others have identified MMPs as the primary effectors of fibroblast-mediated collagenolysis (20, 25, 26). Interestingly, adult mouse fibroblasts express at least six MMPs that can potentially degrade type I collagen, raising the possibility of multiple compensatory networks that are designed to preserve collagenolytic activity (25). Four of these collagenases belong to the family of secreted MMPs, i.e. MMP-13, MMP-8, MMP-2, and MMP-9, whereas the other two enzymes are members of the membrane-type MMP subgroup, i.e. MMP-14 (MT1-MMP) and MMP-16 (MT3-MMP) (13, 27–29). From a functional perspective, the specific roles that can be assigned to secreted versus membrane-anchored collagenases remain undefined. As such, fibroblasts were isolated from either wild-type mice or mice harboring loss-of-function deletions in each of the major secreted and membrane-anchored collagenolytic genes, and the ability of the cells to degrade type I collagen was assessed. Herein, we demonstrate that fibroblasts mobilize either secreted or membrane-anchored MMPs to effectively degrade type I collagen in qualitatively and quantitatively distinct fashions. However, under conditions where fibroblasts use either secreted and membrane-anchored MMPs to exert quantitatively equivalent collagenolytic activity, only MT1-MMP plays a required role in supporting a collagen-invasive phenotype. These data establish a new paradigm wherein secreted collagenases are functionally limited to bulk collagenolytic processes, whereas MT1-MMP uniquely arms the fibroblast with a focalized degradative activity that mediates subjacent collagenolysis as well as invasion.