Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis

Abstract

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

Mice with Hepatic Loss of the Desmosomal Protein γ-Catenin Are Prone to Cholestatic Injury and Chemical Carcinogenesis

“…23 Pkp3 has been shown to promote plaque formation and cell adhesion 24,25 and can also inhibit tumor progression and metastasis. 26 Thus, b-cateninePkp3/Dsg2 complexes in KO may be a result of junctional intermingling, suggesting an important role of g-catenin in proper junctional sorting, as has been shown in global g-catenin KO with cardiac defects. 27 Although the compensations in the absence of g-catenin appear sufficient to maintain desmosome structure and function at baseline, more indepth studies will be essential to address the primary role of proteins, like Dsg2 and Pkp3, in liver pathophysiology and in the context of g-catenin loss.…”

“…33 In various tumors, loss of desmosomal proteins is associated with enhanced tumorigenesis or tumor metastasis. Loss of Pkps is associated with increased tumor cell invasion in colorectal cancers 26 and prostate adenocarcinoma. 34 Loss of Dsc-2 is associated with enhanced tumorigenicity of colorectal cancer through bcatenin activation.…”

14-3-3γ meditated transport of plakoglobin to the cell border is required for the initiation of desmosome assembly in vitro and in vivo

“…The antibodies against PKP3, both DSC2 and DSC3, DSG2, plakoglobin, desmoplakin, K8 (keratin 8), actin, E-cadherin, b-catenin and a-E-catenin were used in western blots as previously described (Gosavi et al, 2011;Khapare et al, 2012;Kundu et al, 2008). Tissue culture supernatants of the antibodies against HA (12CA5), 14-3-3c (CG31) and 14-3-3s (CS112) were used at a dilution of 1:50.…”

“…In some experiments, cells were fixed in 4% paraformaldehyde and permeabilized with Triton X-100 as described previously (Gosavi et al, 2011). The antibodies against PKP3, both DSC2 and DSC3, DSG2, plakoglobin, desmoplakin, K8, actin, E-cadherin, b-catenin, ZO-1 and a-E-catenin were used in immunofluorescence analysis as described previously (Gosavi et al, 2011;Khapare et al, 2012;Kundu et al, 2008). Antibodies against PKP2 (BD Clontech, dilution 1:25), KIF5B (Abcam, dilution 1:100), a-tubulin (Abcam, dilution 1:150), Par3 (Millipore, dilution 1:50), ZO-1 (Abcam, dilution 1:100), P-cadherin (BD Transduction Laboratories, dilution 1:100), HA (12CA5, supernatant), p120 catenin (BD Transductions, dilution 1:100), N-cadherin (Life Technologies, catalog number 33-3900, dilution 1:50), a-E-catenin (Santa Cruz Biotechnology, dilution 1:25) and E-cadherin (clone 36/E-cadherin, mouse monoclonal, BD Transduction Laboratories, dilution 1:100) were incubated with the cells for 1 hour at room temperature at the indicated dilutions as described previously (Gosavi et al, 2011).…”

14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein

“…Immunofluorescence and Confocal Microscopy-Immunofluorescence with antibodies against plakoglobin, desmocollin2/3, desmoglein2, HA, plakophilin3, desmoplakin, ZO1, E-cadherin, ␤-catenin, ␣-tubulin, and keratin8 were performed as described (66,69,70). Vimentin (Sigma; dilution, 1:500) or N-cadherin (BD Transduction Laboratories; dilution, 1:10) were immunostained using methanol fixation as described (66).…”