Receptor protein-tyrosine phosphatase RPTPmu binds to and dephosphorylates the catenin p120(ctn)

RPTPmu is a prototypic receptor-like protein-tyrosine phosphatase (RPTP) that mediates homotypic cell-cell interactions. Intracellularly, RPTPmu consists of a relatively large juxtamembrane region and two phosphatase domains, but little is still known about its substrate(s). Here we show that RPTPmu associates with the catenin p120(ctn), a tyrosine kinase substrate and an interacting partner of cadherins. No interaction is detectable between RPTPmu and beta-catenin. Furthermore, we show that tyrosine-phosphorylated p120(ctn) is dephosphorylated by RPTPmu both in vitro and in intact cells. Complex formation between RPTPmu and p120(ctn) does not require tyrosine phosphorylation of p120(ctn). Mutational analysis reveals that both the juxtamembrane region and the second phosphatase domain of RPTPmu are involved in p120(ctn) binding. The RPTPmu-interacting domain of p120(ctn) maps to its unique N terminus, a region distinct from the cadherin-interacting domain. A mutant form of p120(ctn) that fails to bind cadherins can still associate with RPTPmu. Our findings indicate that RPTPmu interacts with p120(ctn) independently of cadherins, and they suggest that this interaction may serve to control the tyrosine phosphorylation state of p120(ctn) at sites of cell-cell contact.     

Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.     

Identification of Src phosphorylation sites in the catenin p120ctn

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.     

Isolation and characterization of calmodulin genes from Xenopus laevis.

Two cDNAs derived from Xenopus laevis calmodulin mRNA have been cloned. Both cDNAs contain the complete protein-coding region and various lengths of untranslated segments. The two cDNAs encode an identical protein but differ from each other by 5% nucleotide substitutions. The 5' and 3' untranslated regions, to the extent available, are highly homologous between the two cDNAs. The predicted sequence of X. laevis calmodulin is identical to that of vertebrate calmodulins from mammals and chickens and shows one substitution compared with electric eel calmodulin. Genomic DNA sequences homologous to each of the two cDNA clones have been isolated and were shown to account for the major calmodulin-coding DNA sequences in X. laevis. These data suggest that X. laevis carries two active, nonallelic calmodulin genes. Although no complete analysis has been carried out, it appears that the X. laevis calmodulin genes are interrupted by at least four introns. The relative concentrations of calmodulin mRNA have been estimated in different embryonic stages and adult tissues and found to vary by up to a factor of 10. The highest levels of calmodulin mRNA were found in ovaries, testes, and brains. In these three tissues, the two calmodulin genes appear to be expressed at approximately equal levels.     

Isolation and characterization of a monosialosylgangliopentaosyl ceramide from Xenopus laevis oocyte.

A monosialosylgangliopentaosyl ceramide was isolated from Xenopus laevis oocytes. It represented 5.8% of the total acidic glycosphingolipids. From the results of sugar-composition analysis, enzymatic hydrolysis, permethylation analysis, and negative ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows: [sequence: see text] The predominant species of fatty acids were alpha-hydroxy fatty acids, h22:0, h24:0, and h24:1. The long chain bases of this ganglioside consisted mainly of d18:1 sphingosine and phytosphingosine. Other acidic glycolipids were also characterized. The most abundant component of acidic glycolipids was sulfatide, which represented 85.7% of the total acidic glycolipid mixture. GM3, GM2, GM1a, and GD1a were also detected.     

Isolation and characterization of the hatching enzyme from the amphibian,Xenopus laevis

The proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its ¹²⁵I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn²⁺ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn²⁺ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.     

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.     

Isolation and characterization of a lectin from the cortical granules of Xenopus laevis eggs

A cortical granule lectin was isolated from eggs of the South African clawed toad Xenopus laevis. The lectin was released from the cortical granules by activation of dejellied eggs with the Ca2+ ionophore A23187. The lectin was purified by affinity chromatography with its natural ligand, the egg jelly coat, chemically coupled to a Sepharose matrix. The purified lectin was homogeneous by the criteria of isoelectric focusing (pI = 4.6), immunodiffusion, and immunoelectrophoresis but existed in two different molecular weight isomers as determined by sedimentation velocity ultracentrifugation and disc gel electrophoresis. Molecular weights of the isomers were determined by ultracentrifugation, disc gel electrophoresis, and gel filtration and found to be 539,000 and 655,000. Chemically, the lectin was a metalloglycoprotein, composed of 84.0% protein, 15.8% carbohydrate, and 0.19% calcium. No unusual types or amounts of amino acids were present. The carbohydrate moiety was composed of fucose, mannose, galactose, glucosamine, galactosamine, and sialic acid. The monosaccharide specificity of the lectin was investigated with the sugar inhibition of the precipitin reaction in gels. The lectin was specific for D-galactosyl sugars with the configuration at carbon atoms 2-4 of primary importance.     

Isolation, characterization, and extra-embryonic secretion of the Xenopus laevis embryonic epidermal lectin, XEEL

The Xenopus laevis embryonic epidermal lectin (XEEL) is a novel member of a group of lectins including mammalian intelectins, frog oocyte cortical granule lectins, and plasma lectins in lower vertebrates and ascidians. We isolated the XEEL protein from the extract of tailbud embryos by affinity chromatography on a galactose-Sepharose column. The XEEL protein is a homohexamer of 43-kDa N-glycosylated peptide subunits linked by disulfide bonds. It requires Ca(2+) for saccharide binding and shows a higher affinity to pentoses than hexoses and disaccharides. HEK-293T cells transfected with an expression vector containing the XEEL cDNA secrete into the culture medium the recombinant XEEL (rXEEL) that is similar to the purified XEEL in its molecular nature and saccharide-binding properties. Substitution of Asn-192 to Gln removed the N-linked carbohydrate and inhibited secretion of rXEEL but did not abolish the activity to bind to galactose-Sepharose. The embryo's XEEL content, as estimated by western blot analyses, increases during neurula/tailbud stages and declines after 1 week postfertilization. Immunofluorescence and immuno-electron microscopic analyses showed localization of the XEEL protein in a typical secretory granule pathway of nonciliated epidermal cells. When tailbud embryos were cultured in the standard medium, XEEL was accumulated in the medium, indicating secretion of XEEL into the environmental water. The rate of XEEL secretion greatly increased at around the hatching stage and stayed at a high level during the first week after hatching. XEEL may have a role in innate immunity to protect embryos and larvae against pathogenic microorganisms in the environmental water.     

The catenin, p120ctn, is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues

The cadherin-binding catenin p120ctn was originally identified as an Src-tyrosine kinase substrate. More recently, p120ctn has been shown in some cell types to be associated with catenin/cadherin complexes of adherens junctions. To address the question whether p120ctn is restricted to certain cell types or whether it is a general cellular component we investigated tissue distribution of p120ctn by immunohistochemistry and immunoblotting in the rat. We found p120ctn to be widely distributed in several tissues where it is mainly restricted to the plasma membrane. In various epithelia p120ctn was found in association with different adherens junctions such as the zonula adherens and puncta adherentia. In addition, p120ctn was localized along infoldings of the basal cell membrane, most prominently in renal proximal and distal tubules. pl20ctn was not restricted to epithelia. It was also found at intercalated discs of cardiomyocytes. In the nervous system, immunostaining was particularly prominent in areas rich in synapses suggesting that pl20ctn is a component of synaptic adherens junctions as well. By immunoblotting, four different isoforms of pl20ctn could be detected displaying similar electrophoretic mobilities as the isoforms 1A, 1B, 2A, and 2B reported from mice. Whereas all epithelia assayed contained at least two isoforms, testis, heart, brain, and retina contained a single 110-kDa band that corresponds to isoform 1B in mice.     

Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.

p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.     

Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.     

The Human DF3/MUC1 carcinoma-associated antigen signals nuclear localization of the catenin p120(ctn)

The human DF3/MUC1 glycoprotein is aberrantly overexpressed by carcinoma cells. The present studies show that MUC1 associates with the Armadillo protein, p120(ctn). The cytoplasmic domain of MUC1 binds directly to p120. The functional significance of the MUC1-p120 association is supported by the demonstration that MUC1 induces nuclear localization of p120. These findings demonstrate that MUC1 confers cell membrane to nuclear signaling by interactions with p120.     

Isolation of desmosomes from the epidermis of Xenopus laevis and immunochemical characterization of the Xenopus desmosomal cadherins

Here we report a new method of isolating epidermal desmosomes from Xenopus laevis, and a major constituent of desmosomes designated as Xenopus desmogleins (XDsg). Isolation of desmosomes from Xenopus laevis epidermis was carried out by a two step-incubation with different concentrations of NP-40. After discontinuous sucrose gradient centrifugation at 30,000 g for 60 min, a pure desmosomal fraction was obtained at 30%/40% interface. In the SDS-PAGE of isolated desmosomes, at least 12 bands (XDB1 to XDB12) were observed over a 75 kD region. Among them, three bands (XDB3, XDB7, XDB8; estimated MW 175, 124, and 112 kD respectively) were recognized as glycoproteins based on ConA binding. Monospecific polyclonal antibody against XDB3 cross-reacted with bovine Dsgs and vis-a-vis anti-bovine Dsgs with XDB3. By contrast, monospecific antibody against bovine Dsc a/b did not cross-react with either XDB7 or XDB8. Heterogeneous molecular constituents of desmosomal adhesion molecule, which have been observed among different bovine tissues, were confirmed in a phylogenetically different animal, Xenopus laevis. Combined results with other evidence could suggest an alternative system for desmosome-mediated cell adhesion.     

Isolation and characterization of a 7 S RNP particle from mature Xenopus laevis oocytes

Mature oocytes of Xenopus laevis contain a 7 S RNP particle consisting of two components, ribosomal 5 S RNA and a protein of Mr approximately 45000. The structure of the free 5 S rRNA and the 7 S RNP complex has been studied by diethylpyrocarbonate modification of adenines. A74, A77, A90, A100, A101 and A103 of the 5 S rRNA are protected upon association of the protein.