Biochemical characterization of the soluble form of the junctional plaque protein, plakoglobin, from different cell types

Abstract

A polypeptide of identical molecular mass ( M , 83 000) and charge to desmosomal plakoglobin from bovine snout epidermis was identified in soluble and pelletable fractions from diverse tissues and cells of different mammalian species, including cells and tissues devoid of desmosomes (e. g. endothelial, retinal, lenticular cells, fibroblasts). The protein, however, wdS not detected in erythrocytes and platelets and in myeloma cells, nor in smooth muscle tissue. In all cells examined, the plakoglobin soluble upon cell lysis in buffers of near-physiological pH and ionic strength (21 -31 % of the total plakoglobin in the different cell types) was found to exist in a distinct molecular form. On sucrose gradient centrifugation it appeared at about 7 S and gel filtration chromatography revealed a Stokes radius of about 5.0 nm, from which an M , of about 170000 was estimated. By using isoelectric focusing under non-denaturing conditions, soluble z 7-S plakoglobin had an isoelectric point at about pH 5.3. The plaque-bound and the soluble form of plakoglobin were indistinguishable by electrical charge and molecular mass, regardless of the source, indicating molecular identity. Cross-linking of soluble proteins with cupric 1,lOphenanthroline resulted in the formation of a cross-linked product of plakoglobin with similar physical properties as the native z 7-S particle, which is compatible with the interpretation that the soluble plakoglobin particle is a dimer. While a major proportion of the plakoglobin in the desmosomal plaque was resistant to various extraction procedures, plakoglobin present in the plaques of non-desmosome-containing cells and tissues was readily extractable under low and high salt conditions. This indicates that differences exist in the binding of plakoglobin to desmosomal plaques and the plaques of non-desmosomal junctions.

Organization of desmosomal plaque proteins in cells growing at low calcium concentrations.

“…Proteins of the 100,000 g supernatant fraction described above were concentrated by vacuum dialysis against near-physiological salt buffer to a final volume of 500 Ixl (33,34,60). These samples were then loaded on top of 'x,l 1 ml of a 5-30% (wt/vol) continuous sucrose gradient and centrifuged in a Beckman SW40 rotor at 35,000 rpm for 18 h at 5°C.…”

Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells.

“…At the molecular level, it has been shown that, although the two subfamilies share some molecular constituents (i.e., vinculin, a-actinin, and actin), there are some components that are specific for each of them. The cytoplasmic plaque molecule talin (Burridge 1983a,b) and the integrin ECM receptors (Damsky et al, 1985) were shown to be specific for cell-matrix AJ, whereas the plaque molecule plakoglobin (Kapprell et al, 1987) and the transmembrane receptors of the cadherin family (Takeichi, 1988) are uniquely associated with cell-cell AJ. Still poorly understood are the regulatory mechanisms that control AJ formation and stability.…”

Plakoglobin Is O-Glycosylated Close to the N-terminal Destruction Box

“…Therefore, modification of plakoglobin and ␤-catenin appears to be dependent on the cell context. Epidermis has many layers of non-proliferating cells that contain a significant cytosolic fraction of plakoglobin (62). Similarly 293T cells do not proliferate following transient transfection and the expressed plakoglobin is mostly cytoplasmic because of lack of cell junctions in this cell line (11).…”