| Kruppel-like factors (KLFs), the zinc finger containing protein family, play an important role in cell proliferation, apoptosis, cell differentiation, angiogenesis, lymphogenesis, tumorigenesis, as well as in embryonic stem cell (ES cell) development through the regulation of various rich GCs gene promoters Each member of the KLFs family has its unique biological functions and more than 17 family members have been identified. A lot of evidence showed that the abnormal expression of KLFs is correlated with tumorigenesis. However, the detailed mechanisms remain unknown. Recently, KLF2, KLF4, and KLF5 have become the research focus within the KLFs family, because of their functions in cell differentiation, self-renewal, adipocyte differentiation, and tumorigenesis. KLF5 was related to adipocyte, stem cells differentiation and self-renewal. Moreover, it has been regarded as an oncoprotein which is able to promote tumor cell proliferation. KLF2, another member of KLFs, is involved in T cell egress, inhibits adipocyte differentiation, and regulates the self-renew and proliferation of ES cells. KLF2,4, and 5 deficient ES cells lose their totipotency and ability of self-renew. Yet, most studies focus on the transcriptional regulation of the KLFs. Their post-transcriptional modulation remains to be largely unknown.SCFFbw7, an E3 ubiquitin ligase, has a critical role in tumorigenesis, cell proliferation and cell differentiation. This protein complex consists of Fbw7, SKP1, and CUL1 and mediates the degradation of its target protein via ubiquitination dependent pathway. Fbw7, also called FBXW7, CDC4, Sel10, Ago, one of the F-box proteins plays a critical role in identifying its substrates. Fbw7 conserves from yeast to mammalian. Fbw7 degrades its substraes through a conserved concensus CPD(CDC4 phospho-degron):S/TPXXT/D/E. More evidence suggest that Fbw7, an important tumor suppressor, plays a vital role in cell proliferation, differentiation and cell growth. For example, Fbw7 is able to degrade c-Myc, Cyclin E, Notch and c-Jun and these targets are highly related to cell proliferation. Furthermore, clinical samples from bowel, lung and breast cancer, also contain Fbw7 mutations and accumulation of c-Myc and c-Jun. However, the specific molecular mechanisms through which Fbw7 functions as a tumor inhibitor need to be unveiled in future research. Study of mechanism will provide a novel theoretical idea for tumor treatment.In this article, we identified tumor suppressor Fbw7 as a novel E3 ligase for KLF5 and KLF2 using bioinformatics, cytobiology and molecular biology techniques. We further studied the mechanism by which Fbw7 mediates the degradation of KLF5.The major parts of our research are as following:1. Fbw7 regulates the stabilities of KLF5 and KLF2 1) Through the analysis of KLFs family proteins using Scansite software, we idenfied the CPD:S/TPXXT/D/E, a classical signal for Fbw7 dependent degradation in KLF2 and 5.2) To study the degradation of KLF5 by exogenous Fbw7, we overexpressed KLF2, KLF4, KLF5 and Fbw7α, p-TrCP1, Fbw2, Fbw5, Fbw8 in HEK293T cell. Our data showed that only Fbw7a degraded KLF2 and 5, and MG132 treatment inhibited the degradation. This result was confirmed by flow cytometre. Together, our data indicated that Fbw7αdegrade KLF2 and 5 via proteasome degradation pathway.3) To study the degradation of KLF5 by endogenous Fbw7, we transfected siFbw7 into Hela cells to knockdown endogenous Fbw7. Our data showed knocking down Fbw7 up-regulated of KLF5 protein levels, while had minor effect on KLF5 mRNA levels. Moreover, knockdown of Fbw7 delayed the turnover of endogenous KLF5 via the pulse chase assay. The protein levels of KLF5 in Fbw7-deficient HCT116 or DLD1 cells are much higher than their parent cells.Taken together, our data clearly showed that both endogenous and exogenous Fbw7 can efficiently degrade KLF5 and 2.2. The molecular mechanisms of the degradation of KLF2 and 5 by Fbw7.Next, we focused on the molecular mechanisms by which Fbw7 mediated KLF2 and 5 degradation.1) Immunoprecipitation and Pull Down assays were used to detect the interaction between Fbw7 and KLF2 and 5 in vivo and in vitro.2) To identify the domains of interaction between KLF2,5 and FBW7, different mutations were transfected into HEK293T and our data indicated that the T227, S292, T312 sites of KLF5 and the T243, S247 sites of KLF2 are the potential sites for interaction.3) We tested whether Fbw7 promotes KLF2 and KLF5 ubiquitination using an in vivo and in vitro ubiquitination assay. The results showed that Fbw7 can promote the ubiquitination of KLF2 and 5. Knocking down of Fbw7 inhibited the KLF5 ubiquitination.All the above showed that Fbw7 regulated the degradation of KLF2 and 5 via Ubiquitin-proteasome pathway.3. GSK3p is required for the Fbw7-mediated degradation of KLF5.1) KLF5 is able to be phosphorylated by GSK3P in vitro phosphorylation experiment.2) Overexpression of GSK3βcould promote the degradation of KLF5 and knockdown of GSK3βdelayed the turnover of endogenous KLF5 via the pulse chase experiment.3) To further understand the mechanism by which GSK3βaffects KLF5 stability, we examined the effect of GSK3βactivity on the interaction between Fbw7 and KLF5. Treatment with LiCl clearly decreased the interaction between endogenous Fbw7 and KLF5 and also significantly inhibited the ubiquitination of endogenous KLF5Our data showed that GSK3βcan promote the degradation of KLF5. 4. Fbw7-mediated degradation regulates KLF5 biological activity1) Fbw7 inhibits KLF5-promoted cell proliferation. Overexpression of KLF5 promoted cell proliferation. We tested whether Fbw7-mediated degradation inhibited KLF5-dependent cell proliferation using a Soft Agar formation assay. KLF5, Fbw7 or its CPD mutation was transfected into HCT-116 cells. Compared to empty vector, more colonies were formed when wild-type KLF5 was overexpressed, whereas the Fbw7-resistant mutant KLF5-3A was able to stimulate HCT116 cells to form colonies more than the wild type. In addition, co-expression of Fbw7significantly inhibited KLF5-mediated cell proliferation, but had little effect on KLF5-3A-mediated proliferation. Furthermore, The results of MTT also showed that expression of mouse KLF5-3A had a stronger effect on cell proliferation than did the wild type.2) Fbw7 inhibits KLF5 transcriptional activity. Based on our results thus far, we speculated that Fbw7 might inhibit the expression of KLF5-transactivated genes. We therefore measured by RT-PCR the expression levels of the known KLF5-targeted genes in wild-type or Fbw7-deficient HCT116 and DLD1 cancer cells. Survivin mRNA levels in particular were elevated significantly in both types of Fbw7-deficient cells. Subsequent luciferase reporter assays showed that the expression of KLF5 significantly enhanced the activity of the survivin essential promoter in HCT116 cells, whereas this effect was inhibited efficiently by co-expression of Fbw7.Thus, Fbw7 appears to downregulate the transcriptional activity of KLF5 and inhibits KLF5-promoted the colorectal cancer cells proliferation.Taken together, we have identified the novel substrates KLF5 and KLF2 of E3 ligase, Fbw7, via the tools of bioinformatics, cytobiology and molecular biology. For the first time we revealed that the tumor suppressor Fbw7 can down-regulate KLF5 and KLF2 on their protein level. Our work contributes some new clues and methods for the understanding of KLF5 and KLF2 protein stability and biological function. This research identified a novel substrate KLF5 of E3 ligase Fbw7 and the molecular mechanism of the degradation of KLF5 by Fbw7. In conclusion, Fbw7 could promote the degradation of KLF5 via Ubiquitin-proteasome, inhibit the expression of KLF5 downstream genes and the colorectal cancer cells proliferation. KLF5 plays a very important role in tumorigenesis. This new regulatory mechanism of KLF5 degradation may provide significant plateform, where we can learn useful diagnostic and therapeutic targets for tumor and efficacious methods for identifying the new substrates of Fbw7. |
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