title: Identification of the regions of PECAM-1 involved in beta- and gamma-catenin associations.
abstract: Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine-phosphorylated beta-catenin and modulates beta-catenin localization and sequestration. The biological significance of this interaction, while still unclear, it has been postulated to be involved in modulating adherens junction dynamics in response to perturbants [J. Clin. Invest. 109 (2002) 383]. Here we demonstrate that tyrosine-phosphorylated beta-catenin, and to a lesser extent unphosphorylated beta-catenin, interact with a portion of the cytoplasmic domain of PECAM-1 encoded by exon 15. Using RT-PCR, we obtained products representing alternatively spliced PECAM-1 isoforms from mouse kidney total mRNA and generated PECAM-1-GST constructs expressing full length and naturally occurring alternatively spliced PECAM-1 variants. Co-precipitation assays revealed that the protein sequence encoded by exon 15 is necessary for beta-catenin binding. Transfections using deletion mutants confirmed the importance of the exon 15 sequence in this interaction. In contrast, gamma-catenin-PECAM-1 interactions are thought to be modulated by an as yet undefined PECAM-1 serine phosphorylation and appear to mediate dynamic PECAM-1 intermediate filament cytoskeletal interactions [J. Biol. Chem. 275 (2000) 21435]. Here we demonstrate that the PECAM-1-gamma-catenin interaction occurs via an exon 13-mediated process. GST-pull-down assays illustrated the importance of the exon 13 sequence in this interaction. Further, using site-directed mutagenesis of S(673) to C and D and S(669 and 670) to C, we confirmed the importance of S(673) and its phosphorylation state as a mediator of gamma-catenin-PECAM-1 binding. Our studies define the exons of the PECAM-1 cytoplasmic domain that is involved in mediating these PECAM-1-catenin family member interactions and will allow investigators to better define the biological functions resulting from these interactions.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA. Purba_Biswas@brown.edu
authors.forename: Purba
authors.initials: P
authors.lastname: Biswas
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Schoenfeld
authors.forename: David
authors.initials: D
authors.lastname: Schoenfeld
authors.forename: Dita
authors.initials: D
authors.lastname: Gratzinger
authors.forename: Sandra
authors.initials: S
authors.lastname: Canosa
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Madri
mesh_terms: D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D051180:Desmoplakins; D005091:Exons / Q000235:genetics; D006801:Humans; D010768:Phosphoserine / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin; D051185:gamma Catenin
chemical_list: C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; C495292:JUP protein, human; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins; D015534:Trans-Activators; D051176:beta Catenin; D051185:gamma Catenin; D010768:Phosphoserine