| ObjectiveTo observe the effect that PAK2 phosphorylate proto-oncogene protein c-Jun on cell proliferation and transformation though cell internal and cell external experiment, clarify the role of PAK2 in tumor occurrence and development, explore the possibility That PAK2 will be a new target for cancer treatmen and offer new ideas and methods for cancer treatment.Methods1. Intracellular protein expression levels were detected by western blot.2. PAK2 knockdown was done in mouse skin epithelial JB6 C141 cells and melanoma SK-MEL-5 cells by sh-RNA, which impact were observed on cell proliferation, transformation and AP1 activity.3. The effects on cell proliferation of different processing factors were observed in cell proliferation assay.4. Using soft-agar cloning assay (anchorage-independent cell transformation assay) analyzed the impact of PAK2 knockdown to cell transformation in JB6 cells and SK-MEL-5 melanoma cells and stable transfection pcDNA4-mut-c-Jun plasmids to JB6 cells on cell transformation.5. Application of AP1 activity assay to observe how PAK2 knockdown effects cell AP1 activity in JB6 cells and SK-MEL-5 melanoma cells; How stable transfection mutant-c-Jun (5A) plasmids to JB6 cells effects AP-1 activity; How transient transfection PAK2 and c-Jun, PAK2 and mut-c-Jun (5A) plasmids to JB6 cells affects AP1 activity.6. Application of mammalian two-hybrid assay to observe whether PAK2 can combine c-Jun in vitro.7. Using PAK2 kinase assay test whether PAK2 can bind and phsphorylate full-length c-Jun protein in vitro. Test whether PAK2 can phsphorylate full-length c-Jun protein at Ser or Thr.8. Using immunoprecipition assay to observe whether exogenous PAK2 and c-Jun can combine in the cells.9. Phosphorylation threonine sites of c-Jun by PAK2 were detected using c-Jun peptide mapping.Results1. Intracellular protein expression levels were detected by western blot:â‘ EGF can activate PAK2, phosphorylation of PAK2 can be measured after EGF treatment 15 minutes, the phosphorylation activity level showed a gradual increase tread with the extension of stimulation time, and the strongest phosphorylation activity appeared at 3 hours. ERK1/2 phosphorylation by EGF is used as a positive control.â‘¡Some protein expression level were examined in sh-mock and sh-PAK2 stable JB6 cell line by EGF stimulation. The results showed that in PAK2 knockdown stable cell line, all the detected protein expression levels (p-ERK1/2, ERK1/2, p-RSK, RSK, p-c-Jun(Ser63), p-c-Jun(Ser73), c-Jun, c-Fos) did not change compared with sh-mock stable JB6 cell line.â‘¢.wt-c-Jun and mut-c-Jun (5A) plasmids were transfected into JB6 C141 cells to obtain stable JB6 cell line, xpress tags were detected in JB6 cells to make sure that wt-c-Jun and mut-c-Jun (5A) have been successfully transfected.â‘£PAK2 protein expression level were examined in JB6 C141 cells, Hacat cells, melanoma SK-MEL-5 and SK-MEL-28 cells by western blot and found that PAK2 is highly expressed in melanoma SK-MEL-5 and SK-MEL-28 cells.2. PAK2 knockdown was done by sh-PAK2 in mouse skin epithelial JB6 C141 cells and melanoma SK-MEL-5 cells.â‘ .Infection of sh-pak2 stable blocked the endogenous PAK2 protein expression level about 90% compared with sh-mock control JB6 cells.â‘¡.Endogenous PAK2 protein level was suppressed about 80% by sh-PAK2 compared with sh-mock control in melanoma SK-MEL-5 cells.3. The effects on cell proliferation of different processing factors were observed in cell proliferation assay.â‘ .Cell proliferation of sh-PAK2 in jb6 cells was clearly slower than that of sh-mock(p<0.05).â‘¡. Cell proliferation of mut-c-Jun in jb6 cells was clearly slower than that of wt-c-Jun (5A) in jb6 cells (p<0.05).â‘¢.SK-MEL-5 cells with stably infected sh-PAK2 suppress proliferation compared with the sh-mock control (p<0.05).4.Soft-agar cloning assay (anchorage-independent cell transformation assay).â‘ .Infection of sh-PAK2 into JB6 cells generated less colony in soft agar compared with JB6 cells infected sh-mock(p<0.05).â‘¡Transfection of mut-c-Jun (5A) into JB6 cells generated less colony in soft agar compared with JB6 cells transfected wt-c-Jun(p<0.05).â‘¢. sh-PAK2 stable cell line displayed about 60% reduction in soft agar without EGF inducement compared with sh-mock control in SK-MEL-5 cells (p<0.05).5. AP-1 activity assay. â‘ AP-1 activity reduced in JB6 cells stably Infected sh-PAK2 compared with JB6 cells infected sh-mock (p<0.05).â‘¡AP-1 activity reduced in JB6 cells Transfection of mut-c-Jun (5A) compared with JB6 cells transfected wt-c-Jun (p<0.05).â‘¢. AP-1 activity reduced in sh-PAK2 stable cell line compared with sh-mock control in SK-MEL-5 cells (p<0.05).6. Mammalian two-hybrid assay. When pBIND-PAK2 and pACT-c-Jun were co-transfected, the luciferase activity was showed approximately 35 times highter than that of the control group only transfected with pG51u. The co-transfection group of pBIND-JNK1 and pACT-c-Jun is considered as a positive control, which was showed about 65 times higher compared with the control.7. PAK2 kinase assay.â‘ . PAK2 could phosphorylate c-Jun in vitro.â‘¡PAK2 stronger phosphorylated c-Jun at threonine sites and don't phosphorylated serine sites including two well-known sites of c-Jun ser63 and ser73.8. Immunoprecipition assay. V5 tagged PAK2 was combined and precipitated and xpress tagged c-Jun protein be coimmunoprecipitated with PAK2.9. c-Jun peptide mapping.â‘ . T2, T8, T89, T93, T286 five Thr. sites had relatively strong signals, which suggested that these five Thr. sites of c-Jun are possible PAK2 phsphorylation c-Jun Thr Sites. Whereas there is no or weak signal appeared in the remaining three sites T91, T126 and T231, it meant that this three sites cannot be phosphorylated by PAK2.â‘¡. PAK2 phosphorylating c-Jun signals still existed when two, three or four Thr sites were mutant together. And only five, sites were all mutant simultaneously, PAK2 cannot phosphorylate c-Jun. So this indicated that these five Thr sites of c-Jun are exactly phosphorylated by PAK2.Conclusion1. Cell proliferation of sh-PAK2 JB6 cells and SK-MEL-5 cells were slower than that of sh-mock. PAK2 knockdown inhibited JB6 cell and SK-MEL-5 cells transformation in soft agar and AP-1 luciferase activity.2. After c-Jun is mutate to 5A, the transforming ability decreased in JB6 cells, and AP-1 activity reduced in JB6 cells transfection of mut-c-Jun (5A).3. PAK2 can bind and phosphorylate c-Jun at 5 Thr sites, affecting AP-1 activity and thereby promoting cell proliferation and transformation.4. PAK2 can bind and phosphorylate c-Jun at 5 Thr sites. PAK2 can affect AP-1 activity by binding and phosphorylating c-Jun at 5 Thr sites, rather than Ser63 and Ser73.5. PAK2 might be a new target for the clinical chemotherapeutic of malignant tumor in the future.
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