The Role And Mechanism Study Of Phosphorylation Of TOPK At Y74, Y272 By Src In Carcinogenesis Of Colon Caner

Objective:T-LAK cell-originated protein kinase (TOPK), a serine/threonine protein kinase, is highly expressed in a variety of tumors and associated with a poor prognosis of human malignancies. However, the activation mechanism of TOPK is still unrevealed. Src activity increases in 80% of colon cancer patients, and there exsists the Src consensus substrate motif in TOPK. To explore if Src could phosphorylate TOPK directly in colon cancer, and then to vertify the phosphorylation sites and prepare the phosphorylation antibodies. Furthermore, to study the role and mechanism of phosphorylation of TOPK at Y74, Y272 by Src in carcinogenesis of colon caner.Methods:1. An in vitro kinase assay was performed in the presence of [γ-32P] ATP with Src as an active kinase and TOPK as a substrate. The potential tyrosine phosphorylation sites of TOPK were predicted by NetPhos 2.0. Five high-score peptides were then designed, synthesized commercially and incubated with active Src in the presence of [γ-32P] ATP in an in vitro kinase assay.2. P-TOPK (Y74) and p-TOPK (Y272) were prepared. And the wild type (His-TOPK) (WT), Y74F TOPK (74F), Y272F TOPK (272F) and Y74Y272FF TOPK (His-TOPK) (FF) were purified from E coli respectively, and used as substrates for active Src in an in vitro kinase assay.3. That if Src and TOPK could co-localize in SW480 cells was checked under the confocal microscope. Ni-NTA-His-TOPK was used to pull down endogenous Src, and then Src was probed with anti-Src by Western blot.4. PcDNA3-HA-TOPK and pcDNA4-His-Src were cotransfected into 293T cells, and the phosphorylation of TOPK at Y74 was analyzed by p-TOPK (Y74).5. The phosphorylation level of TOPK at Y74 in SW480 cells at 0,5,15 or 30 minutes after EGF treatment, in three different colon cancer cell lines treated with Dasatinib and in shMock- and shSrc-expressing cells were tested.6. TOPK mutants Y74F, Y272F or Y74Y272FF was constructed and the stable cell lines in JB6 and SW480 were set up. Growth curves and the anchorage-independent colony formation ability of stable cell lines was tested.7. The levels of phospho-Histone H3 (p-Histone H3 (S10)) in JB6 stable cell lines, in three different colon cancer cell lines treated with Dasatinib and in shMock-and shSrc-expressing cells were tested.8. The ability of SW480/WT cells to form tumors in athymic nude mice was compared with that of SW480/FF cells and hematoxylin & eosin (H&E) staining and immunohistochemical (IHC) analysis was did after the tumors of mice injected with SW480/WT cells grew to 1000 mm3 and the mice were euthanized.9. His-Src, Flag-ubiquitin or HA-TOPK were cotransfected into HEK293T cells. Then cell extracts from each group were immunoprecipitated with anti-HA, and Flag-ubiquitin were detected by Western blot.10. Subsequetly the half-life of TOPK in 293T cells transiently transfected with pcDNA3-HA-TOPK wild type (TOPK-WT) and pcDNA3-HA-TOPK double mutant (TOPK-FF) and in Src+/+ and Src-/- MEFs were tested.Results:1. The data of in vitro kinase assay indicated that Src could phosphorylate TOPK in vitro; The potential five tyrosine phosphorylation sites of TOPK were predicted by NetPhos 2.0; Src phosphorylated TOPK at Y74 or Y272 in peptide mapping.2. The results of western blot using prepared p-TOPK (Y74) antibody showed that Src could phosphorylate TOPK at Y74, and the signal disappeared in the single mutant TOPK-74F and the double mutant TOPK-FF. These data suggested that Src did phosphorylate TOPK at Y74 in vitro.3. The result showed that Src (red) co-localized with TOPK (green) in both the cytoplasm and nucleus of SW480 cells; The results of western blot indicated that Src could directly bind with TOPK.4. The phosphorylation level of TOPK at Y74 was increased compared to the level of controls when cotransfected with Src and TOPK, and the level was dramatically increased after being stimulated by EGF.5. Endogenous phosphorylation of TOPK at Y74 was increased after EGF treatment; Endogenous phosphorylation level of TOPK at Y74 was gradually decreased in a dose-dependent manner after dasatinib treatment and in shMock-and shSrc-expressing cells.6. The results showed that the growth of 74F,272F, or FF cells in JB6 or SW480 stable cell lines were slower than that of WT cells and the colonies formed by JB6 or SW480/74F, JB6 or SW480/272F and JB6 or SW480/FF cells were significantly fewer and smaller compared with JB6 or SW480/WT group. Mutated TOPK at Y74F, Y272F, or Y74Y272FF could block the anchorage-independent growth ability ex vivo.7. The level of p-Histone H3 (S10) dramatically decreased in JB6/FF cells, in in Src knockout (Src-/-) cells and in the three colon cancer cells with 50nM dasatinib treatment.8. Tumor growth curves indicated that the increasing growth rate in the mice injected with SW480/FF cells was significantly lower than that in the mice inoculated with SW480/WT cells. The result of H&E staining suggested both of them were poorly differentiated adenocarcinoma cells. IHC analysis of the tumor samples from two groups of mice showed that p-Histone H3 (S10) was more highly expressed in SW480/WT cells than that in SW480/FF cells. The tumorigenic properties of SW480/FF cells significantly reduced.9. The overexpression of Src dramatically reduced the ubiquitination level of TOPK.10. TOPK-FF cells had shorter half-life than that of TOPK-WT; The half-life of TOPK in Src+/+cells was much longer than that in Src-/- cells. The phosphorylation of TOPK by Src enhances the stability of TOPK.Conclusion:The phosphorylation of TOPK at Y74 and Y272 by Src increases the stability and activity of TOPK, and promotes the tumorigenesis of colon cancer.