Limits...
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: 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.Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation.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

High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.A. Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. 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 K8 (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. A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. D. Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. E. Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. F. HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368841&req=5

pone-0038561-g001: High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.A. Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. 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 K8 (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. A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. D. Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. E. Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. F. HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.

Mentions: Previous work from this laboratory has demonstrated that PKP3 loss in HCT116 cells leads to increased neoplastic progression and metastasis [33]. PKP3 has been shown to form a complex with K18 in a yeast two hybrid assay [27]. To determine if PKP3 forms a complex with K18 in HCT116 cells, protein extracts from HCT116 cells were incubated with either non-specific IgG or antibodies to K18. The immunoprecipitation reactions were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K18 and PKP3. As shown in Figure S1A, complexes immunoprecipitated by the K18 antibody contain K18 and PKP3, in contrast to extracts incubated with the control antibody. As K8 and K18 are a keratin pair expressed mostly in simple epithelia [9], [10], the levels of K8 and K18 were determined in the HCT116 derived PKP3 knockdown clones S9 and S10 [33]. S9 and S10 were generated using two different shRNA constructs, both show similar defects in desmosome formation, and both show increased transformation and metastasis when injected into nude mice [28], [33]. Hence, all future experiments were performed with these two clones. A Western blot analysis demonstrated that the levels of both K8 and K18 were elevated in the PKP3 knockdown clones (S9 and S10) as compared to the vector control cells (pTU6) (figure 1A). Western blots for actin served as loading controls and a Western blot for PKP3 shows the decrease in PKP3 levels in S9 and S10. A high salt extraction shown in Figure S1B demonstrated that in addition to K8 and K18 one additional band was present that was recognized by an antibody that recognises most type I keratins (data not shown). The PKP3 knockdown clones, S9 and S10, showed an elevation of all three keratins (Figure S1B). The increased keratin levels did not affect filament formation by K8 or K18 (Figure S1C). The other band was not another intermediate filament protein like vimentin as a Western blot demonstrated that either vector control or knockdown cells derived from HCT116 cells do not express vimentin (Figure S1D) as previously reported [34], [35].


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)

High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.A. Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. 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 K8 (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. A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. D. Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. E. Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. F. HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368841&req=5

pone-0038561-g001: High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.A. Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. 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 K8 (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. A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. D. Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. E. Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. F. HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.
Mentions: Previous work from this laboratory has demonstrated that PKP3 loss in HCT116 cells leads to increased neoplastic progression and metastasis [33]. PKP3 has been shown to form a complex with K18 in a yeast two hybrid assay [27]. To determine if PKP3 forms a complex with K18 in HCT116 cells, protein extracts from HCT116 cells were incubated with either non-specific IgG or antibodies to K18. The immunoprecipitation reactions were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K18 and PKP3. As shown in Figure S1A, complexes immunoprecipitated by the K18 antibody contain K18 and PKP3, in contrast to extracts incubated with the control antibody. As K8 and K18 are a keratin pair expressed mostly in simple epithelia [9], [10], the levels of K8 and K18 were determined in the HCT116 derived PKP3 knockdown clones S9 and S10 [33]. S9 and S10 were generated using two different shRNA constructs, both show similar defects in desmosome formation, and both show increased transformation and metastasis when injected into nude mice [28], [33]. Hence, all future experiments were performed with these two clones. A Western blot analysis demonstrated that the levels of both K8 and K18 were elevated in the PKP3 knockdown clones (S9 and S10) as compared to the vector control cells (pTU6) (figure 1A). Western blots for actin served as loading controls and a Western blot for PKP3 shows the decrease in PKP3 levels in S9 and S10. A high salt extraction shown in Figure S1B demonstrated that in addition to K8 and K18 one additional band was present that was recognized by an antibody that recognises most type I keratins (data not shown). The PKP3 knockdown clones, S9 and S10, showed an elevation of all three keratins (Figure S1B). The increased keratin levels did not affect filament formation by K8 or K18 (Figure S1C). The other band was not another intermediate filament protein like vimentin as a Western blot demonstrated that either vector control or knockdown cells derived from HCT116 cells do not express vimentin (Figure S1D) as previously reported [34], [35].

Bottom Line: 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.Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation.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