Localization and binding of WT-JMD and p120-catenin.

<p>(A) Immunofluorescence of MDCK cells transiently expressing ActA or WT-JMD. Images for RFP (red), p120-catenin (green) and merged are shown separately (100×). Boxed areas are shown as higher magnifications below (RFP-, p120- and merge-inset). All images were from the same experiment and processed identically between cell lines. Scale bar is 25 µm in 100× images, and 5 µm in insets. (B) Lysates and RFP immunoprecipitates (IP) of ActA and WT-JMD stable cell lines under normal conditions, upon proteasome inhibition, or NEM treatment (to inhibit de-ubiquitinating enzymes). Immunoblots (IB) for RFP show: a slower migrating band (upper band-JMD-Ub) that appears only in WT-JMD cells in the presence of MG-132 and NEM; the band identified as ActA/HC comprises a co-migrating ActA and the IgG heavy chain (HC). Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of RFP intensities normalized to tubulin in WT-JMD stables cell lines. Data averaged from 3 independent experiments (+/− s.e.m.), and 2 independently cloned stable cell lines; *p≤0.05, **p≤0.01.</p

E-cadherin JMD is Ubiquitinated.

<p>(A) Lysates of WT-JMD stable cell lines under normal conditions, upon proteasome inhibition (MG-132), or inhibition of deubiquitinating enzymes (NEM). Immunoblot (IB) for RFP shows a slower migrating band (JMD-Ub) in the presence of MG-132 and NEM. (B) In a separate experiment MDCK cells stably expressing WT-JMD were transiently transfected with Ub-HA where indicated. Cells were extracted and RFP immunoprecipitations were preformed under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), addition of the deubiquitinating enzyme Usp2, or mock transfections. DTT was added to the immunoprecipitates after the final was of Protein A beads but prior to the addition of Usp2, where indicated, to neutralize residual NEM. Immunoblots (IB) were performed for RFP using a rabbit polyclonal antibody, followed by a sequential immunoblotting with antibodies specific for: 1) tubulin; and 2) HA. The slowest migrating band (marked as JMD-Ub) that appears in the presence of NEM is positive for RFP and HA (lanes 10 and 11). HC denotes IgG heavy chain. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (C) Extracts from MDCK cells stably expressing WT-JMD were immunoprecipitated (IP) for RFP and E-cadherin under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), or addition of the de-ubiquitinating enzyme Usp2. DTT was added to the immunoprecipitates after the final wash of Protein A beads but prior to the addition of Usp2, where indicated, to neutralize residual NEM. A slow migrating band (lane 15) and protein smear (lanes 11, 13, and 14) appear in E-cadherin immunoprecipitates in the presence of NEM, and collapse upon incubation with Usp2 (similar to RFP immunoprecipitates). RFP immunoblots in lanes 11, 13, 14 & 15 show that the slower migrating band does migrates at different molecular weights indicating variable levels of JMD ubiquitination. Number(s) on the side of the blots are the apparent molecular weights of protein standards (× 10^∧3). Data are representative of 3 different experiments.</p

E-cadherin JMD Level is Regulated by Proteasomal Degradation.

<p>(A) Top: schematic representation of the Juxtamembrane domain (JMD) expression construct (left) and ActA control construct (right). Bottom: mouse E-cadherin JMD sequences aligned to demonstrate differences in mutations utilized in this study. Lysine to arginine mutations are denoted by rectangular boxes (K#R and Red “R”). Red AAA denotes mutations to abolish E-cadherin JMD/p120-catenin binding. E-cadherin octapeptide sequence required for binding p120-catenin is underlined. Numbers above the sequence represent the amino acid position number, and the corresponding amino acid position in murine E-cadherin sequence in parenthesis. (B) MDCK cells stably expressing ActA or WT-JMD were incubated for 6 hours with cycloheximide to determine protein turnover. β-catenin used as a degradation control and GAPDH as a loading control. Number(s) on the side of gels represent apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of immunoblots of MDCK cells stably expressing ActA or WT-JMD in cycloheximide time course experiment (see B). Band intensities normalized to GAPDH to account for the reduction in overall protein levels. Results were averaged (+/− standard error of the mean (s.e.m.)) from 3 different experiments (N = 3). (D) Fluorescence images of MDCK cells transiently expressing RFP (ActA) or E-cadherin JMD RFP fusion protein (WT-JMD) targeted to the mitochondria; boxed regions in “RFP” are shown below at a higher magnification in “RFP-inset.” Scale bar is 25 µm in 100X images, and 5 µm in insets. (E) WT-JMD levels increase 4-fold upon proteasome inhibition. WT-JMD migrated with an apparent molecular weight that was ∼15kDa greater than ActA. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (F) Quantification of immunoblot RFP protein levels averaged (+/− s.e.m.) from 3 different experiments; *p≤0.034.</p

Lysine mutations inhibit WT-JMD degradation and differentially affect WT-JMD stability.

<p>(A) RFP immunofluorescence of MDCK cells transiently expressing K5R-JMD, K83R-JMD, or K5,83R-JMD under normal conditions (left column), or upon proteasome inhibition (+ MG-132). Images were processed under identical conditions and from the same experiment as images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037476#pone-0037476-g001" target="_blank">Figure 1D</a>. Scale bar is 25 µm. (B) MDCK cells were transiently transfected with ActA or K5,83R-JMD and incubated for 6 hours with cycloheximide, 40 hours post-transfection. Immunoblots (IB) were performed for RFP, β-catenin and GAPDH. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of immunoblots from MDCK cells transiently expressing ActA or K5,83R-JMD and incubated for 6 hours with cycloheximide, 40 hours post-transfection. Band intensities from post nuclear supernatants were normalized to GAPDH to account for reduction in overall protein levels. Results are averaged from 3 independent experiments (+/− s.e.m.).</p

Expression of Src-GFP or Hakai-FLAG increase JMD degradation and ubiquitination.

<p>(A) MDCK cells stably expressing WT-JMD were transiently transfected with Src-GFP. Cells were extracted and RFP immunoprecipitations (IP) were performed under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), or addition of the de-ubiquitinating enzyme Usp2. De-ubiquitinating enzyme Usp2 (with DTT) was added, where indicated, to immunoprecipitates after the last step of washing Protein A beads. Membranes were immunoblotted (IB) for RFP, GFP, p120-catenin, and tubulin. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (B) Quantification of immunoblots for WT-JMD upon expression of Src-GFP in WT-JMD stable cells. RFP WT-JMD band intensities from immunoprecipitates were normalized to tubulin to account for reduction in overall protein levels as a result of transfections. *p<0.05. Results are averaged from 3 different experiments (+/− s.e.m.). (C) MDCK cells stably expressing WT-JMD were transiently transfected with Hakai-FLAG. Cells were extracted and RFP immunoprecipitations (IP) were preformed under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), or addition of the de-ubiquitinating enzyme Usp2. De-ubiquitinating enzyme Usp2 (with DTT) was added, where indicated, to immunoprecipitates after the last step of washing Protein A beads. Immunoblots (IB) were performed for RFP, p120-catenin and tubulin. Numbers on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (D) Quantification of the ratio of JMD-Ub level to total mitochondrial targeted JMD protein (WT-JMD + JMD-Ub) upon expression of Hakai-FLAG in WT-JMD stable cells. Band intensities from immunoprecipitates were normalized to tubulin. Results are averaged from 3 independent experiments (+/− s.e.m.). *p<0.01, **p<0.001.</p

Analysis of full length E-cadherin lysine mutants verifies results of RFP-JMD mitochondrial degradation assay.

<p>(A) Parental MDCK cells and MDCK cells transiently expressing full length E-cadherin RFP fusion proteins (WT-FL), or full length E-cadherin RFP fusion proteins containing lysine mutant sequences (K5R-FL; K83R-FL; K5,83R-FL; AAA-K5,83R-FL). RFP immunoprecipitates were performed 40 hours post-transfection under normal conditions. Immunoblots (IB) for RFP show higher levels of K5R-FL, K5,83R-FL and AAA-K5,83R-FL compared to WT-FL. Immunoblots for p120-catenin show that p120-catenin is co-immunoprecipitated with all E-cadherin mutants, except AAA-K5,83R-FL. (B) Quantification of levels of transiently expressed E-cadherin RFP fusion proteins in MDCK cells. RFP band intensities were normalized to tubulin. Results are averaged from 3–4 different experiments as indicated (+/− s.e.m.); ^∧ p<0.03, *p<0.01, **p<0.001, ***p<0.0001. (C) RFP immunofluorescence of MDCK cells transiently mock transfected, or expressing WT-JMD-FL, K5R-WT-FL, K83R-FL, K5,83R-FL or AAA-K5,83R-FL. All images were taken in cells in which the proteasome was inhibited (+MG-132) to ensure visualization of RFP in WT-JMD-FL and K83R-FL transiently expressing cell lines. Images were taken and processed under identical conditions to each other. Scale bar is 25 µm.</p

Lysine mutations differentially affect p120-catenin localization.

<p>(A-D) MDCK cells transiently expressing K5R-JMD. (A), K83R-JMD (B), K5,83R-JMD (C) or AAA-K5,83R-JMD (D). Cells were fixed and processed for immunofluorescence for RFP and p120-catenin. Boxed areas are shown at higher magnification below (inset). All images were from the same experiment and processed identically to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037476#pone-0037476-g005" target="_blank">Figure 5A</a>. Scale bar is 25 µm in 100x images, and 5 µm in insets. (E) MDCK cells transiently expressing ActA, WT-JMD, K5,83R-JMD or AAA-K5,83R-JMD. Lysates and RFP immunoprecipitates (IP) were performed 40 hours post-transfection under normal conditions or upon proteasome inhibition. Immunoblots (IB) for RFP show similar levels of ActA, K5,83R-JMD and AAA-K5,83R-JMD levels. Immunoblots for p120-catenin show that p120-catenin is co-immunoprecipitated with K5,83R-JMD (lanes 15, 16) but not AAA-K5,83R-JMD (lanes 13, 14). Number(s) on the side are the apparent molecular weights of proteins standards (× 10^∧3). Quantification of RFP immunoblots from cell lines transiently expressing ActA, WT-JMD, K5,83R-JMD or AAA-K5,83R-JMD. RFP levels for ActA, K5,83R-JMD and AAA-K5,83R-JMD were higher than WT-JMD. Data are averaged from 3 independent experiments (+/− s.e.m.); *p  = 0.031, **p<0.005, ***p≤0.001. (F) RFP immunofluorescence of MDCK cells transiently expressing AAA-K5,83R-JMD under normal conditions or upon proteasome inhibition (+MG-132). Images were taken and processed under identical conditions to each other and those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037476#pone-0037476-g001" target="_blank">Figure 1D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037476#pone-0037476-g004" target="_blank">Figure 4A</a>. Scale bar is 25 µm. (G) MDCK cells stably expressing ActA or WT-JMD were transfected with either scrambled siRNAs (-) or p120-catenin specific siRNAs (+). Cells were extracted and RFP immunoprecipitations were preformed under normal conditions or upon proteasome inhibition (MG-132). Immunoblots (IB) were processed for RFP, p120-catenin and tubulin. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). Quantification of WT-JMD levels upon knockdown of p120-catenin was normalized to tubulin. Results are averaged from 3 independent experiments (+/− s.e.m.); *p<0.05, **p<0.01.</p