Protocadherin of the liver, kidney, and colon associates with detergent-resistant membranes during cellular differentiation

Abstract

Protocadherin LKC (PLKC) is a member of the heterogeneous subgroup of protocadherins that was identified and described as a potential tumor-suppressor gene involved in contact inhibition (Okazaki, N., Takahashi, N., Kojima, S., Masuho, Y., and Koga, H. (2002) Carcinogenesis 23, 1139-1148 and Ose, R., Yanagawa, T., Ikeda, S., Ohara, O., and Koga, H. (2009) Mol. Oncol. 3, 54-66). Several aspects of the structure, posttranslational processing, targeting, and function of this new protocadherin are still not known. Here, we demonstrate that the expression of PLKC at the apical membrane domain and its concentration at regions of cell-cell contacts occur concomitantly with significant elevation of PLKC-mRNA levels. Furthermore, it can be found within the adherens junctions, but it does not colocalize with tight junctions proteins ZO-1 and occludin, respectively. Additionally, unlike E-cadherin, PLKC is not redistributed upon Ca(2+) removal. Biosynthetic labeling revealed N- and O-glycosylation as posttranslational modifications as well as a fast transport to the cell surface and a low turnover rate. During differentiation, PLKC associates with detergent-resistant membranes that trigger its redistribution from intracellular membranes to the cell surface. This association occurs concomitant with alterations in the glycosylation pattern. We propose a role for PLKC in the establishment of a proper epithelial cell polarity that requires O-linked glycosylation and association of the protein with detergent-resistant membranes.

FIGURE 1.

PLKC expression is affected by cell-cell contacts in MDCK-II. A, MDCK-PLKC-YFP cells were plated at low density, and subcellular distribution was analyzed using confocal laser microscopy during formation of a confluent monolayer. Blue arrow, cell surface; white arrow, Golgi apparatus; red arrow, cell-cell contact sites. B, shown is confocal laser microscopy of MDCK-PLKC-YFP cells transiently transfected with cDNA Golgi-dsRed. C, mRNA samples from MDCK-II cells of distinct stages of differentiation were estimated for expression of PLKC-mRNA by semiquantitative PCR. For quantification purposes the β-actin mRNA levels were used. D, Northern blot analysis is shown of polar and non-polar MDCK-II cells including 28 S and 18 S RNA controls. Scale bar, 10 μm.

FIGURE 2.

PLKC is targeted in epithelial cells to the adherens but not to the tight junctions. MDCK-PLKC-YFP cells were grown to confluence and fixed with paraformaldehyde. Cells were permeabilized with 0.5% saponin. A, E-cadherin was stained with anti-E-cadherin antibody. An Alexa Fluor® 568 conjugate was used as secondary antibody. B, shown is an xz-section scan of MDCK-PLKC-YFP cells stained for E-cadherin. C, ZO-1 and occludin were labeled in MDCK-PLKC-YFP cells using a Cy3-conjugated secondary antibody. D, shown is an xz-section scan of MDCK-PLKC-YFP (green) cells stained for occludin (red). Cell integrity was confirmed by DNA staining (blue). Scale bar, 10 μm.

FIGURE 3.

E-cadherin, but not PLKC, is redistributed upon calcium removal. MDCK-PLKC-YFP cells were grown until 5 days after confluence. Thereafter, calcium was chelated by the addition of EGTA (5 mm final concentration). Samples were fixed at different time points (the 10 min time point is shown in A). For visualization of E-cadherin, cells were processed by immunofluorescence as described above. In B, subcellular localization of PLKC after 30 min of incubation with EGTA was visualized by differential section scanning. A basal cell protrusion is indicated by a white arrow. Scale bar, 10 μm.

FIGURE 4.

Biosynthesis of PLKC. A, MDCK-II cells stably expressing PLKC-YFP were labeled with [³⁵S]methionine for 6 h, and the detergent extracts were processed by immunoprecipitation with anti-GFP antibody. Immunoprecipitates were treated with endo H or PNGase F before SDS-PAGE on 5% slab-gels. In pulse-chase experiments, MDCK-PLKC-YFP cells were labeled for 15 min (B and C) or 2.5 h (D) with [³⁵S]methionine and subsequently chased with nonradioactive culture medium for the indicated time intervals. Thereafter, PLKC-YFP was immunoprecipitated from the detergent extracts of the cells with anti-GFP antibodies. Where indicated, intact cells were treated with 100 μg trypsin/ml in PBS for 45 min on ice before cell lysis.

FIGURE 5.

PLKC associates with DRMs during differentiation. Two-day confluent (A) or subconfluent (∼50%) (B) cells were lysed with 1% (w/v) of Triton X-100 in PBS. After low speed centrifugation, the sample was diluted to a final sucrose concentration of 40% and laid onto an 80% sucrose cushion. The 1-ml sample was then overlaid with 7 ml of 30% sucrose and 1 ml of 5% sucrose on the top and finally centrifuged for 18 h at 100,000 × g with a swing-out rotor. Nine fractions of 1 ml each were collected from the top and analyzed for the protein content by immunoblotting. The distribution of the marker proteins flotillin-2 and RhoA was tested as positive and negative control, respectively. subconf, subconfluent.

FIGURE 6.

Influence of O-glycosylation on PLKC differentiation. A, 2-day confluent or subconfluent (∼50%) MDCK-PLKC-YFP were lysed, and proteins were precipitated, treated with PNGase F or not treated, separated via SDS-PAGE, and visualized by immunoblotting. B, confluent MDCK-PLKC-YFP cells were treated with BNG for 24 h or not treated. Subsequently, cells were lysed, and proteins were precipitated, treated with PNGase F or not treated, separated via SDS-PAGE, and visualized by immunoblotting. C, confluent cells were treated for 24 h with BNG and thereafter lysed with 1% (w/v) of Triton X-100 in PBS. After low speed centrifugation, the sample was diluted to a final sucrose concentration of 40% and laid onto an 80% sucrose cushion. The 1-ml sample was then overlaid with 7 ml of 30% sucrose and 1 ml of 5% sucrose on the top and finally centrifuged for 18 h at 100,000 × g with a swing-out rotor. Nine fractions of 1 ml each were collected from the top and analyzed for the protein content by immunoblotting. sub, subconfluent; conf, confluent. D, the experimental setup was the same as described in C, but the cells were treated with DMM instead of BNG.

FIGURE 7.

Subcellular distribution of PLKC changes after cholesterol depletion or BNG treatment but is independent of N-glycosylation. MDCK-PLKC-YFP cells were plated on coverslips, and subcellular distribution was analyzed using confocal laser microscopy after treatment with BNG, DMM, or methyl-β-cyclodextrin (Cyclo D).