Jack Lilien

Activation of the repulsive receptor Roundabout inhibits N-cadherin-mediated cell adhesion

The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of β-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

Turn‐off, drop‐out: Functional state switching of cadherins

The classic cadherins are a group of calcium dependent, homophilic cell–cell adhesion molecules that drive morphogenetic rearrangements and maintain the integrity of cell groups through the formation of adherens junctions. The formation and maintenance of cadherin‐mediated adhesions is a multistep process and mechanisms have evolved to regulate each step. This suggests that functional state switching plays an important role in development. Among the many challenges ahead is to determine the developmental role that functional state switching plays in tissue morphogenesis and to define the roles of each of the several regulatory interactions that participate in switching. One correlate of the loss of cadherin‐mediated adhesion, the “turn‐off” of cadherin function, is the exit, or “drop‐out” of cells from neural and epithelial layers and their conversion to a motile phenotype. We suggest that epithelial mesenchymal conversions may be initiated by signaling pathways that result in the loss of cadherin function. Tyrosine phosphorylation of β‐catenin is one such mechanism. Enhanced phosphorylation of tyrosine residues on β‐catenin is almost invariably associated with loss of the cadherin‐actin connection concomitant with loss of adhesive function. There are several tyrosine kinases and phosphatases that have been shown to have the potential to alter the phosphorylation state of β‐catenin and thus the function of cadherins. Our laboratory has focused on the role of the nonreceptor tyrosine phosphatase PTP1B in regulating the phosphorylation of β‐catenin on tyrosine residues. Our data suggest that PTP1B is crucial for maintenance of N‐cadherin‐mediated adhesions in embryonic neural retina cells. By using an L‐cell model system constitutively expressing N‐cadherin, we have worked out many of the molecular interactions essential for this regulatory interaction. Extracellular cues that bias this critical regulatory interaction toward increased phosphorylation of β‐catenin may be a critical component of many developmental events.

Continuous association of cadherin with β-catenin requires the non-receptor tyrosine-kinase Fer

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by α- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. We now show that the phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. We have mapped the interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts.

Cables links Robo-bound Abl kinase to N-cadherin-bound β-catenin to mediate Slit-induced modulation of adhesion and transcription

Binding of the secreted axon guidance cue Slit to its Robo receptor results in inactivation of the neural, calcium-dependent cell–cell adhesion molecule N-cadherin, providing a rapid epigenetic mechanism for integrating guidance and adhesion information. This requires the formation of a multimolecular complex containing Robo, Abl tyrosine kinase and N-cadherin. Here we show that on binding of Slit to Robo, the adaptor protein Cables is recruited to Robo-associated Abl and forms a multimeric complex by binding directly to N-cadherin-associated β-catenin. Complex formation results in Abl-mediated phosphorylation of β-catenin on tyrosine 489, leading to a decrease in its affinity for N-cadherin, loss of N-cadherin function, and targeting of phospho-Y489-β-catenin to the nucleus. Nuclear β-catenin combines with the transcription factor Tcf/Lef and activates transcription. Thus, Slit-induced formation of the Robo–N-cadherin complex results in a rapid loss of cadherin-mediated adhesion and has more lasting effects on gene transcription.

PTP1B Modulates the Association of β-Catenin with N-cadherin through Binding to an Adjacent and Partially Overlapping Target Site

The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of β-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872–891 fail to bind PTP1B. This domain partially overlaps with the β-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH2-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the β-catenin target, effectively competes for binding of β-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on β-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the β-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated β-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or β-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with β-catenin, stable expression at the cell surface, and consequently, cadherin function.

Alterations in CDH15 and KIRREL3 in Patients with Mild to Severe Intellectual Disability

Cell-adhesion molecules play critical roles in brain development, as well as maintaining synaptic structure, function, and plasticity. Here we have found the disruption of two genes encoding putative cell-adhesion molecules, CDH15 (cadherin superfamily) and KIRREL3 (immunoglobulin superfamily), by a chromosomal translocation t(11;16) in a female patient with intellectual disability (ID). We screened coding regions of these two genes in a cohort of patients with ID and controls and identified four nonsynonymous CDH15 variants and three nonsynonymous KIRREL3 variants that appear rare and unique to ID. These variations altered highly conserved residues and were absent in more than 600 unrelated patients with ID and 800 control individuals. Furthermore, in vivo expression studies showed that three of the CDH15 variations adversely altered its ability to mediate cell-cell adhesion. We also show that in neuronal cells, human KIRREL3 colocalizes and interacts with the synaptic scaffolding protein, CASK, recently implicated in X-linked brain malformation and ID. Taken together, our data suggest that alterations in CDH15 and KIRREL3, either alone or in combination with other factors, could play a role in phenotypic expression of ID in some patients.

Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination

Mutations in P0 (MPZ), the major myelin protein of the peripheral nervous system, cause the inherited demyelinating neuropathy Charcot-Marie-Tooth disease type 1B. P0 is a member of the immunoglobulin superfamily and functions as a homophilic adhesion molecule. We now show that point mutations in the cytoplasmic domain that modify a PKC target motif (RSTK) or an adjacent serine residue abolish P0 adhesion function and can cause peripheral neuropathy in humans. Consistent with these data, PKCα along with the PKC binding protein RACK1 are immunoprecipitated with wild-type P0, and inhibition of PKC activity abolishes P0-mediated adhesion. Point mutations in the RSTK target site that abolish adhesion do not alter the association of PKC with P0; however, deletion of a 14 amino acid region, which includes the RSTK motif, does abolish the association. Thus, the interaction of PKCα with the cytoplasmic domain of P0 is independent of specific target residues but is dependent on a nearby sequence. We conclude that PKC-mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is necessary for P0-mediated adhesion, and alteration of this process can cause demyelinating neuropathy in humans.

ER-bound PTP1B is targeted to newly forming cell-matrix adhesions.

Here, we define the mechanism through which protein tyrosine phosphatase 1B (PTP1B) is targeted to cell-matrix adhesion sites. Green fluorescent protein (GFP)-labeled PTP1B bearing the substrate-trapping mutation D181A was found in punctate structures in lamellae. The puncta co-localized with focal adhesion kinase (FAK) and Src, and defined the distal tips of cell-matrix adhesion sites identified with paxillin and vinculin. PTP1B is largely associated with the external face of the endoplasmic reticulum (ER) and the puncta develop from ER projections over cell-matrix adhesion sites, a process dependent on microtubules. Deletion of the ER-targeting sequence resulted in cytosolic localization and altered the distribution of PTP1B at cell-matrix foci, whereas mutations disrupting interactions with Src homology 3 (SH3) domains, and the insulin and cadherin receptors had no effect. PTP1B recognizes substrates within forming adhesion foci as revealed by its preferential association with paxillin as opposed to zyxin-containing foci. Our results suggest that PTP1B targets to immature cell-matrix foci in newly forming lamellae by dynamic extensions of the ER and contributes to the maturation of these sites.

Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.

Previously, we demonstrated that chick embryos treated with antisense oligonucleotides against a striated muscle‐specific Xin exhibit abnormal cardiac morphogenesis (Wang et al. [1999] Development 126:1281–1294); therefore, we surmised a role for Xin in cardiac development. Herein, we examine the developmental expression of Xin through immunofluorescent staining of whole‐mount mouse embryos and frozen heart sections. Xin expression is first observed within the heart tube of embryonic day 8.0 (E8.0) mice, exhibiting a peripheral localization within the cardiomyocytes. Colocalization of Xin with both β‐catenin and N‐cadherin is observed throughout embryogenesis and into adulthood. Additionally, Xin is found associated with β‐catenin within the N‐cadherin complex in embryonic chick hearts by coimmunoprecipitation. Xin is detected earlier than vinculin in the developing heart and colocalizes with vinculin at the intercalated disc but not at the sarcolemma within embryonic and postnatal hearts. At E10.0, Xin is also detected in the developing somites and later in the myotendon junction of skeletal muscle but not within the costameric regions of muscle. In cultured C2C12 myotubes, the Xin protein is found in many speckled and filamentous structures, coincident with tropomyosin in the stress fibers. Additionally, Xin is enriched in the regions of cell–cell contacts. These data demonstrate that Xin is one of the components at the adherens junction of cardiac muscle, and its counterpart in skeletal muscle, the myotendon junction. Furthermore, temporal and spatial expressions of Xin in relation to intercalated disc proteins and thin filament proteins suggest roles for Xin in the formation of cell–cell contacts and possibly in myofibrillogenesis.

Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis.

P0, the major peripheral nervous system (PNS) myelin protein, is a member of the immunoglobulin supergene family of membrane proteins and can mediate homotypic adhesion. P0 is an essential structural component of PNS myelin; mice in which P0 expression has been eliminated by homologous recombination (P0−/−) develop a severe dysmyelinating neuropathy with predominantly uncompacted myelin. Although P0 is thought to play a role in myelin compaction by promoting adhesion between adjacent extracellular myelin wraps, as an adhesion molecule it could also have a regulatory function. Consistent with this hypothesis, Schwann cells in adult P0−/− mice display a novel molecular phenotype: PMP22 expression is down‐regulated, MAG and PLP expression are up‐regulated, and MBP expression is unchanged. As in quaking viable mutant mice (qkv), which have uncompacted myelin morphologically similar to that found in P0−/− mice, neither the qKI‐6 or qKI‐7 proteins are expressed in P0−/− peripheral nerve. In addition to these changes in gene expression in the P0 knockout, PLP/DM‐20 accumulates in the endoplasmic reticulum of P0−/− Schwann cells, whereas MAG accumulates in redundant loops of uncompacted myelin, not at nodes of Ranvier or Schmidt‐Lantermann incisures. Taken together, these results demonstrate that P0 is involved, either directly or indirectly, in the regulation of both myelin gene expression and myelin morphogenesis. J. Neurosci. Res. 60:714–724, 2000.