The immunoprecipitates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and -catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type YLF-466D YLF-466D and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion. (Indianapolis, IN) phosphatase assay kit. For the in vitro binding assays to N-cadherin, the GST YLF-466D fusion protein was cleaved with thrombin after binding to the glutathione affinity column (Guan and Dixon, 1991) and then pure recombinant PTP1B was eluted. All constructs were verified by sequencing. Antibodies Chicken-specific polyclonal anti-PTP1B (chkPTP) was prepared in rabbits using a synthetic peptide specific to the chick PTP1B (amino acids 357 to 367; see Fig. ?Fig.11 (Santa Cruz, CA). Enzyme (AP or HRP)-conjugated anti-mouse, -rat, or -rabbit IgG were from Cappel Laboratories (ICN Pharmaceuticals, Costa Mesa, CA). The secondary antibodies conjugated to magnetic beads used in immunoprecipitations were obtained from PerSeptive Diagnostics (Cambridge, MA). Open in a separate window Figure 1 Chick PTP1B cDNA and protein. (for 2.5 h at 4C. The interfaces between YLF-466D 0.25 and 1.2 M sucrose (plasma membrane) and 1.2 and 2 M sucrose (ER) were collected and assayed for protein content using the bicinchoninic acid (BCA) method (and aliquots of the supernatant containing equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylene difluoride (PVDF) membranes. The transfers were immunoblotted with the antibodies indicated in the figures as described previously (Balsamo et al., 1995). Adhesion Assays Cell layers were washed free of serum and harvested using 0.1% trypsin (laser confocal microscope (model LSM 310; and (amino acids 347C356). The antibody recognizes the GST fusion protein (data not shown) and predominant bands at 50 and 40 kD in chick retina homogenates separated by SDS-PAGE (Fig. ?(Fig.11 and and and and and and and [phase]). LN cells expressing mutant GFPCchkPTP1B show little or no fluorescence at the cell periphery (and [phase]) and lack the well-defined borders seen in control cells and cells expressing wild-type GFPCchkPTP1B. Bar, 20 m. Open in a separate window Figure 9 Association of GFPCchkPTP1B fusion protein with N-cadherin. LN cells transfected with GFPC chkPTP1B were homogenized in neutral detergent-containing buffer and immunoprecipitated with antiCN-cadherin antibody NCD-2. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (at 55 kD, position of immunoglobulin heavy chain derived from the antiCc-myc antibody. em Left /em , migration of N-cadherin and PTP1B. em Right /em , numbers indicate the migration of the molecular mass standards. Discussion Our data indicate that PTP1B plays a role in maintaining cadherin in a functional state. This state is characterized by the integrity of the cadherinC-cateninC-cateninCactin connection. Displacement of active PTP1B from the cytoplasmic domain of N-cadherin by transfection of cells with an inactive form of PTP1B results in enhanced tyrosine phosphorylation of -catenin, and loss of its association with cadherin and the actin-containing cytoskeleton. Thus, PTP1B plays an important role in controlling cadherin function by dephosphorylating tyrosine residues on -catenin, and maintaining the cadherinCcytoskeletal linkage. The association of PTP1B with cadherin is regulated by its tyrosine phosphorylation. This, in turn, is regulated by the interaction between a chondroitin sulfate proteoglycan with its cell surface receptor, a cell surface Ctnna1 glycosyltransferase (GalNAcPTase). This interaction initiates a signal which blocks phosphorylation of PTP1B resulting in increased tyrosine phosphorylation of -catenin and loss of cadherin function (Balsamo et al., 1996). PTP1B appears to YLF-466D be involved in many different cellular functions; these diverse functions must depend on specific targeting of the enzyme inside the cell. The enzyme is localized to the cytoplasmic face of the ER via the last 35 amino-acid residues at the COOH terminus (Frangioni et al., 1992). It is cleaved by calpain in response to integrin stimulation in platelets, with concomitant relocation from the ER to the cytosol (Frangioni.