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Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, Gupta N, Alam H, Karkhanis M, Naik N, Vaidya MM, Dalal SN - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Tata Memorial Centre, Kharghar Node, Navi Mumbai, Maharashtra, India.

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.

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Plakophilin3 loss leads to an increase in PRL3 levels.A. RNA prepared from the vector control or PKP3 knockdown clones was used as a template in reverse transcriptase coupled PCR reactions to determine the mRNA levels of PRL3 and PKP3. A PCR for GAPDH served as a loading control. B. S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to PRL3 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) [28]. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. C. The vector control or PKP3 knockdown clones were treated with either the vehicle control (DMSO) or the indicated concentrations of the PRL3 inhibitor. Protein extracts were resolved on gels followed by Western blotting with antibodies to K8 and β-actin. D. GFP PRL3 was transfected into either vector control (pTU6) or PKP3 knockdown clones (S9 and S10). 48 hours post transfection, the cells were stained with antibodies to K8 (red) and visualized by confocal microscopy. Note that GFP PRL3 shows a marginally enhanced localization to the border in S9 and S10 cells in contrast to pTU6 and doesn’t show increased localization on K8 filaments. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. E. Total cell extracts (T), Soluble fractions (S) and the filament fractions (F) were prepared as described from either the vector control or PKP3 knockdown clones. Equal cell equivalents of these extracts were loaded on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8 (top four panels). A Western blot for β-actin was performed in the total cell extracts as a loading control (fifth panel). 100 µg of soluble fractions were resolved on SDS-PAGE gels followed by Western blotting for K8 or β-actin (bottom two panels).
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pone-0038561-g002: Plakophilin3 loss leads to an increase in PRL3 levels.A. RNA prepared from the vector control or PKP3 knockdown clones was used as a template in reverse transcriptase coupled PCR reactions to determine the mRNA levels of PRL3 and PKP3. A PCR for GAPDH served as a loading control. B. S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to PRL3 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) [28]. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. C. The vector control or PKP3 knockdown clones were treated with either the vehicle control (DMSO) or the indicated concentrations of the PRL3 inhibitor. Protein extracts were resolved on gels followed by Western blotting with antibodies to K8 and β-actin. D. GFP PRL3 was transfected into either vector control (pTU6) or PKP3 knockdown clones (S9 and S10). 48 hours post transfection, the cells were stained with antibodies to K8 (red) and visualized by confocal microscopy. Note that GFP PRL3 shows a marginally enhanced localization to the border in S9 and S10 cells in contrast to pTU6 and doesn’t show increased localization on K8 filaments. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. E. Total cell extracts (T), Soluble fractions (S) and the filament fractions (F) were prepared as described from either the vector control or PKP3 knockdown clones. Equal cell equivalents of these extracts were loaded on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8 (top four panels). A Western blot for β-actin was performed in the total cell extracts as a loading control (fifth panel). 100 µg of soluble fractions were resolved on SDS-PAGE gels followed by Western blotting for K8 or β-actin (bottom two panels).

Mentions: It has been reported previously that an increase in PRL3 levels leads to a decrease in K8 phosphorylation and an increase in K8 levels [23]. A Western blot analysis demonstrated that an increase in PRL3 levels was observed in the PKP3 knockdown clones (S9 and S10) as compared to the vector control (pTU6) (figure 1A). This decrease was not due to an increase in mRNA levels as a Reverse Transcriptase coupled polymerase chain reaction (PCR) demonstrated that PRL3 RNA levels were similar in the vector control and PKP3 knockdown clones (figure 2A). As expected PKP3 mRNA levels were lower in the PKP3 knockdown clones as compared to the vector control and GAPDH served as a loading control (figure 2A). To determine if the increase in PRL3 levels could be reversed by an shRNA resistant PKP3 cDNA, a rescue experiment was performed as described above. As shown in figure 2B, S9 cells expressing dsRed PKP3 3.7R showed a decrease in PRL3 staining compared to cells transfected with dsRed. These results suggest that loss of PKP3 leads to an increase in PRL3 protein levels.


Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, Gupta N, Alam H, Karkhanis M, Naik N, Vaidya MM, Dalal SN - PLoS ONE (2012)

Plakophilin3 loss leads to an increase in PRL3 levels.A. RNA prepared from the vector control or PKP3 knockdown clones was used as a template in reverse transcriptase coupled PCR reactions to determine the mRNA levels of PRL3 and PKP3. A PCR for GAPDH served as a loading control. B. S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to PRL3 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) [28]. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. C. The vector control or PKP3 knockdown clones were treated with either the vehicle control (DMSO) or the indicated concentrations of the PRL3 inhibitor. Protein extracts were resolved on gels followed by Western blotting with antibodies to K8 and β-actin. D. GFP PRL3 was transfected into either vector control (pTU6) or PKP3 knockdown clones (S9 and S10). 48 hours post transfection, the cells were stained with antibodies to K8 (red) and visualized by confocal microscopy. Note that GFP PRL3 shows a marginally enhanced localization to the border in S9 and S10 cells in contrast to pTU6 and doesn’t show increased localization on K8 filaments. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. E. Total cell extracts (T), Soluble fractions (S) and the filament fractions (F) were prepared as described from either the vector control or PKP3 knockdown clones. Equal cell equivalents of these extracts were loaded on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8 (top four panels). A Western blot for β-actin was performed in the total cell extracts as a loading control (fifth panel). 100 µg of soluble fractions were resolved on SDS-PAGE gels followed by Western blotting for K8 or β-actin (bottom two panels).
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pone-0038561-g002: Plakophilin3 loss leads to an increase in PRL3 levels.A. RNA prepared from the vector control or PKP3 knockdown clones was used as a template in reverse transcriptase coupled PCR reactions to determine the mRNA levels of PRL3 and PKP3. A PCR for GAPDH served as a loading control. B. S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to PRL3 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) [28]. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. C. The vector control or PKP3 knockdown clones were treated with either the vehicle control (DMSO) or the indicated concentrations of the PRL3 inhibitor. Protein extracts were resolved on gels followed by Western blotting with antibodies to K8 and β-actin. D. GFP PRL3 was transfected into either vector control (pTU6) or PKP3 knockdown clones (S9 and S10). 48 hours post transfection, the cells were stained with antibodies to K8 (red) and visualized by confocal microscopy. Note that GFP PRL3 shows a marginally enhanced localization to the border in S9 and S10 cells in contrast to pTU6 and doesn’t show increased localization on K8 filaments. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. E. Total cell extracts (T), Soluble fractions (S) and the filament fractions (F) were prepared as described from either the vector control or PKP3 knockdown clones. Equal cell equivalents of these extracts were loaded on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8 (top four panels). A Western blot for β-actin was performed in the total cell extracts as a loading control (fifth panel). 100 µg of soluble fractions were resolved on SDS-PAGE gels followed by Western blotting for K8 or β-actin (bottom two panels).
Mentions: It has been reported previously that an increase in PRL3 levels leads to a decrease in K8 phosphorylation and an increase in K8 levels [23]. A Western blot analysis demonstrated that an increase in PRL3 levels was observed in the PKP3 knockdown clones (S9 and S10) as compared to the vector control (pTU6) (figure 1A). This decrease was not due to an increase in mRNA levels as a Reverse Transcriptase coupled polymerase chain reaction (PCR) demonstrated that PRL3 RNA levels were similar in the vector control and PKP3 knockdown clones (figure 2A). As expected PKP3 mRNA levels were lower in the PKP3 knockdown clones as compared to the vector control and GAPDH served as a loading control (figure 2A). To determine if the increase in PRL3 levels could be reversed by an shRNA resistant PKP3 cDNA, a rescue experiment was performed as described above. As shown in figure 2B, S9 cells expressing dsRed PKP3 3.7R showed a decrease in PRL3 staining compared to cells transfected with dsRed. These results suggest that loss of PKP3 leads to an increase in PRL3 protein levels.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Tata Memorial Centre, Kharghar Node, Navi Mumbai, Maharashtra, India.

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.

Show MeSH
Related in: MedlinePlus