Figure 1.
Identification of the protein-protein interaction between TRAPPC4 and ERK2 by co-immunoprecipitation and pull-down assay.
A: Co-immunoprecipitation of TRAPPC4 and ERK2. 293T cells were transfected with empty pcDNA vector or pcDNA vector to express Flag tagged ERK2 (42 kD), or Myc tagged TRAPPC4 (26 kD). Cell lysates were subjected to immunoprecipitation with anti-Flag-HRP antibody or anti-Myc-HRP antibody. The presence of ERK2-Flag and TRAPPC4-Myc in cell extracts prior to immunoprecipitation was controlled using anti-Flag and anti-Myc antibodies (Input). Co-transfection of expression vectors for p16-Myc and TSSK1-Flag was used for the positive control (lane 1). B: Representative results of GST pulldown validation experiments. (a) Immunoblot analysis of GST, and GST-ERK2 proteins used as negative control (lane 2) and bait (lane3) for the pulldown assay. Proteins were detected using anti-6×HIS antibody following SDS-PAGE and membrane transfer. Lane 1 showed the blank control without GST or GST-ERK2. (b) ERK2 expressed as a GST fusion protein from bacteria was bound to beads and incubated with TRAPPC4 expressed as 6×His-TRAPPC4 fusion protein for a pulldown experiment. GST-ERK2 protein(lane1), GST protein(lane2) and TRAPPC4 protein(lane4) were detected by Coomassie staining. Lane3 showed the marker.
Figure 2.
Expression of TRAPPC4 influences the activation status of ERK1/2 in CRC cells.
Cells were lysed, and equal amounts of protein were analyzed by Western blot analysis using antibodies against phospho-ERK1/2 (pERK1/2), or ERK1/2, or TRAPPC4. The density of the Western blot bands were normalized to the amount of GAPDH protein. The data shown are representative of three replicate experiments. *,p<0.05. A. Cells were transfected with TRAPPC4 siRNA (siTRAPPC4) to knockdown TRAPPC4 expression or with Negtive Control siRNA (siControl) as control; Also cells were treated with PDGF to activate ERK-MAPK pathway, or with its solvent (PBS+0.1 BSA) as control. B. Cells were transfected with pCDEF-myc-TRAPPC4 vector to overexpress TRAPPC4 protein or with pCDEF-myc vector as control; Also cells were treated with PDGF to activate ERK-MAPK pathway, or with its solvent (PBS+0.1 BSA) as control.
Figure 3.
Knockdown of TRAPPC4 decreased actived ERK1/2 level in nucleus on SW1116 cell line.
SW1116 cells were transfected with TRAPPC4 siRNA (siTRAPPC4) to knockdown TRAPPC4 expression or with Negtive Control siRNA (siControl) as control; Also cells were treated with PDGF to activate ERK-MAPK pathway, or with its solvent (PBS+0.1% BSA) as control. Cytoplasm and nuclear extracts were prepared as described in experimental procedures, and equal amounts of protein were analyzed by Western blot analysis using antibodies against phospho-ERK1/2 (pERK1/2), or ERK1/2, or TRAPPC4. The density of the Western blot bands were normalized to the amount of GAPDH protein for cytoplasma protein or TBP for nuclear protein. The data shown are representative of three replicate experiments. *,p<0.05.
Figure 4.
Overexpression of TRAPPC4 increased actived ERK1/2 level in nucleus on SW1116 cell line.
SW1116 cells were transfected with pCDEF-myc-TRAPPC4 vector to overexpress TRAPPC4 protein or with pCDEF-myc vector as control; Also cells were treated with PDGF to activate ERK-MAPK pathway, or with its solvent (PBS+0.1% BSA) as control. Nuclear (A) and cytoplasm (B) extracts were prepared as described in experimental procedures, and equal amounts of protein were analyzed by Western blot analysis using antibodies against phospho-ERK1/2 (pERK1/2), or ERK1/2, or TRAPPC4. The density of the Western blot bands were normalized to the amount of TBP for nuclear protein or GAPDH protein for cytoplasma protein. The data shown are representative of three replicate experiments. *,p<0.05.
Figure 5.
Expression of TRAPPC4 inflenced cell growth and its depletion induced apoptosis on SW1116.
A: CCK-8 assay. SW1116 cells were seeded in a 96-well plate until subconfluent. Viable cells were determined by CCK-8 assay. Three independent time were performed in triplicate. Cells were treated with TRAPPC4 siRNA to knockdown TRAPPC4 expression or with Negtive Control siRNA used as control (a), as well as tranfered with pCDEF-myc-TRAPPC4 vector to overexpress TRAPPC4 protein or with pCDEF-myc vector as control (b). *, p<0.05. B: Apoptosis analysis. After SW1116 cells were treated with TRAPPC4 siRNA or Negtive Control siRNA used as control for 48 h. Apoptosis was carried out using the Annexin V assay with Propidium iodide counterstaining allowing quantification by flow cytometry. Three independent time were performed in triplicate. The data were presented as means ± standard deviation (SD). There was significant difference between the two groups( p<0.01).
Figure 6.
Representative immunohistochemical examination of TRAPPC4.
(A), pERK1/2 (B) and ERK1/2 (C) in normal colonic epithelium (a), adenoma (b), and adenocarcinoma (c). Polygrams(d)show expression of the proteins in cytoplasm (pink), nucleus (yellow), and total (blue). A: Expression of TRAPPC4 has no significant difference in the three groups totally, but increases in nucleus from a to c. B: pERK1/2 mainly located in nucleus, and its level increases significantly from a to c. C: Expression of ERK1/2 has no significant difference in the three groups totally, but increases in nucleus from a to c. Original magnification ×200.
Table 1.
Frequency of Expression of TRAPPC4, pERK1/2 and ERK1/2.
Figure 7.
The speculated role of TRAPPC4-ERK2 interaction in CRC.
Extracellular signals, such as PDGF, activate ERK cascades(RAS/RAF/MER/ERK) through receptor tyrosine kinases (RTKs) and utimately regulate cell proliferation and apoptosis. TRAPPC4 binds and activates ERK1/2 as well as participates in the nuclear transport of pERK1/2. When TRAPPC4 is depleted, less ERK1/2 is phosphorylated, and relatively more pERK1/2 remains in the cytoplasm, which leads to suppression of cell proliferation and apoptosis. When TRAPPC4 is overexpressed, more ERK1/2 is activated, and even more is transported into the nucleus, finally leading to increased cell growth.