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PRL-3 promotes cell adhesion by interacting with JAM2 in colon cancer

View Article: PubMed Central - PubMed

ABSTRACT

Phosphatase of regenerating liver-3 (PRL-3), also termed PTP4A3, is a metastasis-related protein tyrosine phosphatase. Its expression levels are significantly correlated with the progression and survival of a wide range of malignant tumors. However, the mechanism by which PRL-3 promotes tumor invasion and metastasis is not clear. In the present study, the functions of PRL-3 were systemically analyzed in the key events of metastasis including, motility and adhesion. A cell wounding assay, cell spread assay and cell-matrix adhesion assay were carried out to analyze the cell movement and cell adhesion ability of colon cancer, immunoprecipitation and immunofluorescence assay was confirmed the interaction of PRL-3 and JAM2. It was demonstrated that PRL-3 promoted the motility of Flp-In-293 and LoVo colon cancer cells and increased the distribution of cell skeleton proteins on the cell protrusions. In addition, stably expressing PRL-3 reduced the spreading speed of colon cancer cells and cell adhesion on uncoated, fibronectin-coated and collagen I-coated plates. Mechanistically, junction adhesion molecular 2 (JAM2) was identified as a novel interacting protein of PRL-3. The findings of the present study revealed the roles of PRL-3 in cancer cell motility and adhesion process, and provided information on the possibility of PRL-3 increase cell-cell adhesion by associating with JAM2.

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PRL3 interacts with JAM2. (A) The candidate interaction protein of PRL-3 in a yeast two hybrid system. (B) Interaction between PRL-3 and JAM2. Cells were cotransfected with PEBG-JAM2, PcDNA3-myc-PRL-3 plasmids and its control. Protein lysates were immunoprecipitated with an anti-myc antibody or GST antibody. Precipitates and protein were analyzed by western blotting with antibodies against GST and myc. (C) Colocalization of PDSRED-JAM2 and GFP-PRL-3 in LoVo cells. Cells were cotransfected with PDSRED-JAM2 and GFP-PRL-3 vector for 48 h. Scale bar, 25 µM. JAM2, junctional adhesion molecular 2; IgG H, IgG heavy chain.
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f3-ol-0-0-4836: PRL3 interacts with JAM2. (A) The candidate interaction protein of PRL-3 in a yeast two hybrid system. (B) Interaction between PRL-3 and JAM2. Cells were cotransfected with PEBG-JAM2, PcDNA3-myc-PRL-3 plasmids and its control. Protein lysates were immunoprecipitated with an anti-myc antibody or GST antibody. Precipitates and protein were analyzed by western blotting with antibodies against GST and myc. (C) Colocalization of PDSRED-JAM2 and GFP-PRL-3 in LoVo cells. Cells were cotransfected with PDSRED-JAM2 and GFP-PRL-3 vector for 48 h. Scale bar, 25 µM. JAM2, junctional adhesion molecular 2; IgG H, IgG heavy chain.

Mentions: To explore the mechanism of PRL-3 in cell adhesion and cell movement, two yeast hybrid systems were used to screen the potential interacting protein(s) of PRL-3 (Fig. 3A). Using BD-PRL-3 fusion protein as a bait protein to screen the embryo brain cDNA librabry, it was demonstrated that JAM2 was a candidate interacting proteins of PRL-3. To confirm the results of the two-yeast hybrid, the interaction between PRL-3 and JAM2 were examined by immunoprecipation with myc-PRL3, followed by western blot analysis with an anti-GST antibody against GST-JAM2,. In addition, GST-JAM2 was pulled down and the precipitate was subjected to western blot analysis using an anti-myc antibody against myc-PRL-3 (Fig. 3B). JAM2 is a known protein located on cell membrane, and PRL-3 also locates on cell membrane and plasma. Plasmids encoding pDsred-JAM2 and pEGFP-GFP-PLR-3 were co-transfected into LoVo cells. After 48 h, the co-localization of exogenous JAM2 and PRL-3 were observed in the cell membrane (Fig. 3C), therefore, PRL-3 may be associated with JAM2 in vitro.


PRL-3 promotes cell adhesion by interacting with JAM2 in colon cancer
PRL3 interacts with JAM2. (A) The candidate interaction protein of PRL-3 in a yeast two hybrid system. (B) Interaction between PRL-3 and JAM2. Cells were cotransfected with PEBG-JAM2, PcDNA3-myc-PRL-3 plasmids and its control. Protein lysates were immunoprecipitated with an anti-myc antibody or GST antibody. Precipitates and protein were analyzed by western blotting with antibodies against GST and myc. (C) Colocalization of PDSRED-JAM2 and GFP-PRL-3 in LoVo cells. Cells were cotransfected with PDSRED-JAM2 and GFP-PRL-3 vector for 48 h. Scale bar, 25 µM. JAM2, junctional adhesion molecular 2; IgG H, IgG heavy chain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4998106&req=5

f3-ol-0-0-4836: PRL3 interacts with JAM2. (A) The candidate interaction protein of PRL-3 in a yeast two hybrid system. (B) Interaction between PRL-3 and JAM2. Cells were cotransfected with PEBG-JAM2, PcDNA3-myc-PRL-3 plasmids and its control. Protein lysates were immunoprecipitated with an anti-myc antibody or GST antibody. Precipitates and protein were analyzed by western blotting with antibodies against GST and myc. (C) Colocalization of PDSRED-JAM2 and GFP-PRL-3 in LoVo cells. Cells were cotransfected with PDSRED-JAM2 and GFP-PRL-3 vector for 48 h. Scale bar, 25 µM. JAM2, junctional adhesion molecular 2; IgG H, IgG heavy chain.
Mentions: To explore the mechanism of PRL-3 in cell adhesion and cell movement, two yeast hybrid systems were used to screen the potential interacting protein(s) of PRL-3 (Fig. 3A). Using BD-PRL-3 fusion protein as a bait protein to screen the embryo brain cDNA librabry, it was demonstrated that JAM2 was a candidate interacting proteins of PRL-3. To confirm the results of the two-yeast hybrid, the interaction between PRL-3 and JAM2 were examined by immunoprecipation with myc-PRL3, followed by western blot analysis with an anti-GST antibody against GST-JAM2,. In addition, GST-JAM2 was pulled down and the precipitate was subjected to western blot analysis using an anti-myc antibody against myc-PRL-3 (Fig. 3B). JAM2 is a known protein located on cell membrane, and PRL-3 also locates on cell membrane and plasma. Plasmids encoding pDsred-JAM2 and pEGFP-GFP-PLR-3 were co-transfected into LoVo cells. After 48 h, the co-localization of exogenous JAM2 and PRL-3 were observed in the cell membrane (Fig. 3C), therefore, PRL-3 may be associated with JAM2 in vitro.

View Article: PubMed Central - PubMed

ABSTRACT

Phosphatase of regenerating liver-3 (PRL-3), also termed PTP4A3, is a metastasis-related protein tyrosine phosphatase. Its expression levels are significantly correlated with the progression and survival of a wide range of malignant tumors. However, the mechanism by which PRL-3 promotes tumor invasion and metastasis is not clear. In the present study, the functions of PRL-3 were systemically analyzed in the key events of metastasis including, motility and adhesion. A cell wounding assay, cell spread assay and cell-matrix adhesion assay were carried out to analyze the cell movement and cell adhesion ability of colon cancer, immunoprecipitation and immunofluorescence assay was confirmed the interaction of PRL-3 and JAM2. It was demonstrated that PRL-3 promoted the motility of Flp-In-293 and LoVo colon cancer cells and increased the distribution of cell skeleton proteins on the cell protrusions. In addition, stably expressing PRL-3 reduced the spreading speed of colon cancer cells and cell adhesion on uncoated, fibronectin-coated and collagen I-coated plates. Mechanistically, junction adhesion molecular 2 (JAM2) was identified as a novel interacting protein of PRL-3. The findings of the present study revealed the roles of PRL-3 in cancer cell motility and adhesion process, and provided information on the possibility of PRL-3 increase cell-cell adhesion by associating with JAM2.

No MeSH data available.


Related in: MedlinePlus